Methods and Compositions for the Display of Polypeptides on the Pili of Gram-Positive Bacteria

ABSTRACT

Provided herein are methods and compositions for the display of polypeptides of interest on the tip of pili of Gram-positive bacteria. According to the present invention, the polypeptide of interest is amino terminal to a Gram-positive bacterial pilus tip protein or an active variant or fragment thereof, wherein the active variant or fragment comprises a cleaved cell wall sorting signal (CWSS) motif. The Gram-positive bacterium displaying a polypeptide of interest on the tip of pili that are disclosed herein are useful, for example, in methods for immunizing a subject with an antigen and methods for removing contaminants from a composition.

FIELD OF THE INVENTION

The present invention relates to the field of microbial polypeptidedisplay.

BACKGROUND OF THE INVENTION

Heterologous surface display of proteins on recombinant microorganismsinvolves the targeting and anchoring of heterologous proteins to theouter surface of host-cells such as yeast, fungi, mammalian and plantcells. Display of heterologous proteins at these cells' surfaces cantake many forms, varying from the expression of reactive groups such asantigenic determinants, heterologous enzymes, (single-chain) antibodies,polyhistidyl tags, peptides, and other compounds. Heterologous surfacedisplay has been applied as a tool for research in microbiology,molecular biology, vaccinology, and biotechnology.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods and compositions for the displayof at least one polypeptide of interest on the tip of pili ofGram-positive bacteria. Methods comprise introducing into aGram-positive bacterium a polynucleotide that encodes a chimericpolypeptide to produce a transformed Gram-positive bacterium expressingthe chimeric polypeptide. The chimeric polypeptide comprises thepolypeptide of interest and a Gram-positive bacterial pilus tip proteinor an active variant or fragment thereof. The pilus tip protein, activevariant or fragment thereof comprises a cell wall sorting signal (CWSS)and is carboxyl to the polypeptide of interest. The Gram-positivebacterium expressing the chimeric polypeptide also expresses a tipsortase and a pilus shaft polypeptide. The transformed Gram-positivebacterium is then grown under conditions wherein the pili are formed.The pili produced by the transformed bacteria display the polypeptide ofinterest.

Compositions comprise Gram-positive bacterium comprising a polypeptideof interest covalently attached to the tip of a pilus, wherein thepolypeptide of interest is amino terminal to a Gram-positive bacterialpilus tip protein or an active variant or fragment thereof, wherein theactive variant or fragment comprises a cleaved cell wall sorting signal(CWSS) motif. The Gram-positive bacterium displaying a polypeptide ofinterest on the tip of pili that are disclosed herein are useful inmethods for immunizing a subject with an antigen, methods for removingcontaminants from a composition (e.g., soil, water), and methods forimproving food products.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the FCT region of serotype M3 Group A Streptococcusstrain M3 and derived constructs. The positions of HA tags and mutationsintroduced by site-specific mutagenesis are indicated by arrowheads.Vectors pCR2.1 and pCR-XL are E. coli cloning vectors (Invitrogen), andpRegP23 (Barnett et al. (2007) J Bacteriol 189:1866-1873), is aGram-positive-E. coli shuttle vector. Zähner and Scott (2008) refers toZähner and Scott (2008) J Bacteriol 190:527-535, which is hereinincorporated by reference in its entirety.

FIGS. 2A-2C show the identification of the Cpa(HA)-T3 dimer in E. colit.Cell lysates of E. coli TOP10 containing plasmid pCR2.1 (lane 1),pJRS1325 (lane 2), and pJRS1326 (lane 3) analyzed:

FIG. 2A presents a Western blot reacted of whole cell lysates withanti-HA antiserum (reproduced from Zähner and Scott (2008) J Bacteriol190:527-535);

FIG. 2B shows the results of immunoprecipitation of a crude extract with“EZview Red Anti-HA Affinity Gel”, followed by boiling in SDS and SDSPAGE stained with SYPRORuby. Monomeric Cpa(HA) (Cpa), the putativeCpa-(HA)-T3 dimer (Cpa-T3), and the light chain (lc) and heavy chain(hc) of IgG are indicated on the right; and

FIG. 2C presents the protein sequence of T3 (SEQ ID NO: 7) with regionscovered by tryptic peptides identified by mass spectrometry indicated inbold. The N-terminal signal peptide (SP) and the C-terminal cell wallsorting signal (CWSS) are underlined.

FIGS. 3A-3D demonstrate the effect of mutations in the CWSS motif ofCpa(HA)

(FIGS. 3A and 3B) and of T3 (FIGS. 3C and 3D) on formation of theCpa(HA)-T3 heterodimer in E. coli:

FIGS. 3A and 3B show a Western immunoblot analysis of hot SDS-treatedcell lysates of E. coli TOP10 containing plasmids pJRS1325 (lane 1),pEU7646 (lane 2), and pEU7904 (lane 3) reacted with (FIG. 3A) monoclonalanti-HA antibody or (FIG. 3B) polyclonal anti-T3 antiserum. The anti-T3antiserum also reacts weakly with Cpa(HA). The locations of the Cpa(HA)monomer (Cpa), the Cpa(HA)-T3 heterodimer (Cpa-T3), and the T3 monomer(T3) and dimer (T3-T3) are indicated on the right; and

FIGS. 3C and 3D show a Western immunoblot analysis of cell lysates of E.coli TOP10 containing plasmids pJRS1325 (lane 1), pEU7646 (lane 2), andpEU7905 (lane 3) reacted with (FIG. 3C) anti-T3 antiserum or (FIG. 3D)anti-HA antibody. The locations of the Cpa(HA) monomer (Cpa), theCpa(HA)-T3 heterodimer (Cpa-T3), and the T3 monomer (T3) and dimer(T3-T3) are indicated. The sizes of molecular mass standards (inkilodaltons) are indicated to the left.

FIGS. 4A and 4B shows that the mutation of the Cpa CWSS motif from VPPTGto VP prevents the formation of the Cpa(HA)-T3 heterodimer andincorporation of Cpa into HMW pilus polymers in GAS.

FIG. 4A shows a Western immunoblot analysis of cell wall extracts (lanes1-4) and 10-fold concentrated supernatants (lanes 5-8) from GAS strainsJRS4/pJRS9545 (lanes 1 and 5), JRS4/pJRS9550 (lanes 2 and 6),JRS4/pJRS9554 (lanes 3 and 7), and JRS4/pJRS9597 (lanes 4 and 8)analyzed with a monoclonal anti-HA antibody.

FIG. 4B shows a Western immunoblot analysis of the same cell wallextracts from JRS4/pJRS9545 (lane 1), JRS4/pJRS9550 (lane 2),JRS4/pJRS9554 (lane 3), and JRS4/pJRS9597 (lane 4) analyzed withpolyclonal anti-T3 antiserum. Molecular masses are indicated to the leftof the figures. The locations of the Cpa monomer (CPa(HA)), the Cpa-T3heterodimer (Cpa(HA)-T3), Cpa linked to a T3 homodimer (Cpa(HA)-(T3)²),Cpa linked to a T3 homotrimer (Cpa(HA)-(T3)³), and the T3 monomer (T3)are indicated on the right of FIGS. 4A and 4B. pJRS9545 is derived frompJRS9508, and consists of the pReg696 backbone and the P23 promoter.(wt): wild type; (vc): vector control

FIG. 5 shows a sequence alignment of the amino acid sequences of Group AStreptococcus major pilin proteins. Strains shown represent thedifferent serotypes containing: FCT-2 (M1), FCT4 (M12, M28), or FCT-3(all others) regions. The M type is indicated followed by the strainname. Invariant lysine residues are indicated in red (K43, K81, K100,K106, K173, and K191). Residues predicted to be involved inintramolecular bond formation are indicated in blue (aspartic acid; N180and N₃O₇) and brown (glutamic acid; E129 and E264). The positionsindicated for K, N, E are deduced from homology to the T3 sequence. Theamino acid sequence of the major pilin protein for M3_MGAS315 strain isset forth in SEQ ID NO: 36; M1_SF370 is SEQ ID NO: 37; M28_MGAS6180 isSEQ ID NO: 38; emmstD33_D633 is SEQ ID NO: 39; M49591 is SEQ ID NO: 40;M18_MGAS8232 is SEQ ID NO: 41; emm33_(—)29487 is SEQ ID NO: 42;MS_Manfredo is SEQ ID NO: 43; and M12_A735 is SEQ ID NO: 44.

FIGS. 6A-C show the effect of replacement of lysine with alanine orarginine in T3 on T3 polymerization in E. coli. Western immunoblotsreacted with polyclonal anti-T3 antiserum.

FIG. 6A shows cell lysates of E. coli TOP10 strains containing plasmid:pEU7655 (lane 1), pEU7657 (lane 2), pEU7678 (lane 3), pEU7679 (lane 4),pEU7680 (lane 5), pEU7681 (lane 6), pEU7682 (lane 7);

FIG. 6B shows E. coli TOP10 strains containing plasmid: pEU7655 (lane1), pEU7657 (lane 2), pEU7678 (lane 3), pEU7682 (lane 4), and pEU7909(lane 5); and

FIG. 6C shows E. coli TOP10 strains containing plasmid: pEU7655 (lane1), pEU7657 (lane 2), pEU7907 (lane 3), pEU7692 (lane 4), and pEU7908(lane 5). The position of the T3 monomer (T3), dimer (T3-T3), and trimer(T3)³ are indicated on the right. The sizes of molecular mass standards(in kilodaltons) are indicated on the left.

FIG. 7 shows the effect of replacement of lysine with alanine orarginine in T3 on T3 polymerization in GAS. A Western immunoblotanalysis of cell wall extracts (lanes 1-4) and supernatants (lanes 5-8)of JRS4/pJRS9536 (lane 1, lane 5), pJRS9541 (lane 2, lane 6), pJRS9543(lane 3, lane 7), pJRS9538 (lane 4, lane 8) reacted with monoclonalanti-HA antibody is shown. The position of the T3 monomer (T3) isindicated on the right. The sizes of molecular mass standards (inkilodaltons) are indicated on the left.

FIG. 8 shows the effect of replacement of lysine with alanine in T3 onCpa(HA)-T3 heterodimer formation in E. coli. Western immunoblots areshown that were reacted with monoclonal anti-HA antibody. FIG. 8A showscell lysates of BL21(DE3)CodonPlus-RIL with pJRS1325 (lane 1), pEU7646(lane 2), pEU7652 (lane 3), pEU7651 (lane 4), pEU7653 (lane 5), pEU7654(lane 6), pEU7661 (lane 7). FIG. 8B shows E. coli TOP10 containingplasmid pEU7646 (lane 1), pEU7687 (lane 2), pEU7688 (lane 3). TheCpa(HA) monomer is labeled Cpa, and the Cpa(HA)-T3 heterodimer islabeled Cpa-T3. The sizes of molecular mass standards (in kilodaltons)are indicated on the left. wt, wild type.

FIGS. 9A and 9B show the effect of replacement of lysine with alanine inT3 on incorporation of Cpa(HA) and T3 polymerization in GAS. Westernimmunoblots are shown with cell wall extracts (lanes 1-5) andsupernatants (lanes 6-9) of JRS4/pJRS9545 (lane 1), JRS4/pJRS9554 (lanes2 and 6), JRS4/pJRS9550 (lanes 3 and 7), JRS4/pJRS9557 (lanes 4 and 8),and JRS4/pJRS9558 (lanes 5 and 9) reacted with monoclonal anti-HAantibody (FIG. 9A) or polyclonal anti-T3 antiserum (FIG. 9B).(vc)=vector control.

FIG. 10 provides photographs of whole-bacteria, negative-staintransmission electron microscopic images of JRS4/pJRS9545 (vectorcontrol; panels A and D) or JRS4/pJRS9550 (Cpa(HA), SipA2, T3, andSrtC2; panels B, C, E, and F). In panels A-C, the bacteria wereincubated with anti-T3 antiserum, followed by an anti-rabbit secondaryantibody conjugated to 12-nm diameter gold particles. In panels D-F, thebacteria were labeled with the anti-T3 antiserum as above, together withanti-HA antibody and an anti-mouse secondary antibody conjugated to18-nm diameter gold particles. The larger gold particles, specific forCpa(HA), could be seen at the tips of the pilus fibers (arrows in panelsE and F). Scale bars=500 nm (panels A-D) or 100 nm (panels E and F).

FIG. 11 illustrates alternative models of Cpa incorporation into the T3pilus structure. In the first model (A), the minor pilin, Cpa, isattached by the VPPTG motif in its CWSS to the α-amino group at theN-terminus of the major pilin, T3. In the second model (B), the VPPTGmotif (SEQ ID NO: 10) in the CWSS of Cpa is attached to K173 of T3 inplace of a T3 subunit. An unknown K residue of Cpa is then used to bondto the QVPTG motif (SEQ ID NO: 9) of the CWSS of T3. This leads to astructure with Cpa interspersed among T3 subunits. In the third model(C), Cpa is linked to a K in T3 other than K173. In the fourth model(D), Cpa can only attach to K173 of T3, and therefore Cpa constitutesthe tip of the pilus.

FIG. 12 shows the expression of MalE/Cpa fusion constructs in E. colistrain XL10. Constructs contain maltose binding protein (MBP)/Cpathrough SrtC2 (pJRS9555) or MBP/Cpa through T3 (pJRS9556). The Westernimmunoblot in panel A was probed with an anti-MBP antibody, while theblot in panel B was probed with an anti-T3 antibody. Lanes 3,6,7 wereconfirmed by PCR to have the desired insert, lanes 2,4,5 lack thisinsert and lanes 8-10 lack srtC2. Lanes:(1) molecular mass standard, (2)pJRS9555.3, (3) pJRS9555.4, (4) pJRS9555.5, (5) pJRS9555.6, (6)pJRS9555.7, (7) pJRS9555.8, (8) pJRS9556.1, (9) pJRS9556.2, (10)pJRS9556.3.

FIG. 13 provides a depiction of the regions encoded by the pJRS9550 andpJRS9565 plasmids.

FIGS. 14A and 14B show the MBP/Cpa fusion protein (MBP*) is surfaceexposed in L. lactis. Whole cell dot blots of L. lactis MG1363containing the plasmid pJRS9565 (lanes 1-4, rows E, F, (+)) or thepJRS9566 plasmid (lanes 1-4, row G, (-SrtC2)) were analyzed with amonoclonal anti-MBP antibody (FIG. 14A) or a polyclonal anti-T3 antibody(FIG. 14B).

FIGS. 15A and 15B shows that the MBP/Cpa fusion protein (MBP*) isincorporated into T3 pili in L. lactis. Western blot analyses areprovided of L. lactis cell wall extracts of MG1363/pJRS9566 (lane3,(-SrtC2)) and MG1363/pJRS9545 (lane 4, (−)) analyzed with a monoclonalanti-MBP antibody (FIG. 15A) or a polyclonal anti-T3 antibody (FIG.15B). Molecular masses are indicated to the left of the figure. Thelocations of the MBP/Cpa fusion protein (MBP*), the MBP-Cpa-T3heterotetramer (MBP*-(T3)³) are shown to the right of the figure.HMW=high molecular mass species

FIGS. 16A-16C provide photographs of immunogold electron microscopy (EM)of MBP*-T3 pili in L. lactis MG1363/pJRS9565. Whole-bacteria,negative-stain transmission EM of MG1363/pJRS9565 (FIGS. 16A and 16B)and MG1363/pJRS9545 (FIG. 16C, vector control) incubated with anti-T3antiserum, followed by an anti-rabbit gold conjugate secondary antibody.

FIGS. 17A and 17B show that the MBP/Cpa fusion protein (MBP*) issynthesized in an active form in L. lactis. Lysates of MG1363/pJRS9545(lane 1, (vc)), MG1363/pJRS9565 (lane 2, (MBP*)) and MG1363/pJRS9566(lane 3, (-SrtC2)) were purified with amylose resin and analyzed with amonoclonal anti-MBP antibody (FIG. 17A) or polyclonal anti-T3 antiserum(FIG. 17B). Molecular masses are indicated to the left of the figure.The locations of the MBP/Cpa fusion protein (MBP*), and the MBP-Cpa-T3heterodimer (MBP*-T3) are shown to the right of the figure.

FIG. 18 shows that T3 pili containing the MBP/Cpa fusion protein (MBP*),but not wild type T3 pili bind to amylose resin. A Western blot analysisis provided of lysates of MG1363/pJRS9565 and MG1363/pJRS9550 that werepurified using amylose resin. Samples corresponding to the eluatefraction of MG1363/pJRS9565 (lanes 1 and 6, (E)), and the eluate (lanes2 and 7, (E)), flow through (lanes 3 and 8, (F)) and crude extract(lanes 4 and 9, (C)) fractions of MG1363/pJRS9550 were analyzed usingmonoclonal anti-MBP (lanes 1-4) or monoclonal anti-HA (lanes 6-9)antibodies. The locations of the MBP/Cpa fusion protein (MBP*), theMBP-Cpa-T3 heterodimer (MBP*-T3), the Cpa(HA) monomer (Cpa), and theCpa(HA)-T3 heterodimer (Cpa-T3) are shown. Molecular masses areindicated to the left of the figure. HMW=high molecular mass species.MM=molecular mass standard.

FIG. 19 provides a graph depicting the levels of anti-MBP IgG in sera ofmice that were intranasally vaccinated with L. lactis MG1363/pJRS9545(control vector) or with MG1363/pJRS9565 (expressing MBP). The barsrepresent an average of ten mice for the experimental groups and twomice for the control group.

FIG. 20 provides a graph depicting the levels of anti-MBP IgA in lunglavage fluid of mice subjected that were intranasally vaccinated with L.lactis MG1363/pJRS9545 (control vector) or with MG1363/pJRS9565(expressing MBP). The bars represent an average of ten mice for theexperimental groups and two mice for the control group.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended embodiments.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a bacterium” is understood to representone or more bacteria. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

Throughout this specification and the embodiments, the words “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

As used herein, the term “about,” when referring to a value is meant toencompass variations of, in some embodiments ±50%, in some embodiments±40%, in some embodiments ±30%, in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

The presently disclosed subject matter provides methods for producingGram-positive bacteria having at least one polypeptide of interestattached to the tip of at least one pilus, wherein the method comprisesintroducing into a Gram-positive bacterium a polynucleotide comprising anucleotide sequence that encodes a chimeric polypeptide. The chimericpolypeptide comprises the polypeptide of interest and a Gram-positivebacterial pilus tip protein, or an active variant or fragment thereof,wherein the pilus tip protein or active variant or fragment thereofcomprises a cell wall sorting signal (CWSS). The chimeric polypeptide isexpressed such that the pilus tip protein, variant, or fragment thereofis carboxyl to the heterologous polypeptide. The transformedGram-positive bacterium additionally expresses a tip sortase and a pilusshaft polypeptide. The transformed Gram-positive bacterium is then grownunder conditions that allow formation of the pilus.

Also disclosed herein are compositions comprising Gram-positive bacteriahaving at least one polypeptide of interest attached to the tip of atleast one pilus. These Gram-positive bacteria find use in methods forinducing an immunological response in a subject through theadministration of a Gram-positive bacterium where the polypeptide ofinterest comprises an antigen. The transformed Gram-positive bacteria ofthe invention also can be used in bioremediation methods, wherein acontaminant is removed from a composition (e.g., soil, water) via theintroduction of a Gram-positive bacterium displaying a biosorbent thatis capable of adsorbing the contaminant or an enzyme that is capable ofdegrading the contaminant. Other uses involve biocatalysis, screeningfor polypeptide expression, the production of biofuels, diagnostics, anduse in probiotics.

Without being bound by any theory or mechanism of action, it is believedthat the presence of the polypeptide of interest on the tip of the piliwill remove the polypeptide further away from the bacterial capsule,enhancing the odds that the polypeptide will fold and function properly.Further, in those embodiments wherein the polypeptide of interestcomprises an antigen, it is believed that displaying the polypeptide onthe tip of the pili will maximize the exposure of the polypeptide to thecells of the immune system, enhancing the immunological responsegenerated against the antigen.

There are two predominant types of bacteria that are categorized basedon the composition and structure of the bacterial cell wall. Whether agiven species of bacteria has one or the other type of cell wall cangenerally be determined by the cell's reaction to certain dyes. Perhapsthe most widely-used dye for staining bacteria is the Gram stain. Whenstained with this crystal violet and iodine stain, bacteria which retainthe stain are called Gram-positive, and those that do not are calledGram negative.

As used herein, by “Gram-positive bacteria” is meant a strain, type,species, or genera of bacteria that, when exposed to Gram stain, retainsthe dye and is, thus, stained blue-purple. The Gram-positive bacterialcell wall contains a relatively thick coat of peptidoglycan.

By contrast, a “Gram-negative bacteria” is meant a strain, type,species, or genera of bacteria that, when exposed to Gram stain does notretain the dye and thus, is not stained blue-purple.

Gram-positive bacteria useful for the presently disclosed methods andcompositions include, but are not limited to, bacteria in the followinggenera: Actinomyces, Bacillus, Bifidobacterium, Cellulomonas,Clostridium, Corynebacterium, Enterococcus, Lactococcus, Lactobacillus,Micrococcus, Mycobactenum, Nocardia, Staphylococcus, Streptococcus, andStreptomyces. In some embodiments, the Gram-positive bacterium isselected from the group consisting of Streptococcus pyogenes,Streptococcus gordinii, Lactococcus lactis, Staphylococcus xylosus, andStaphylococcus carnosus. In particular embodiments, the Gram-positivebacterium comprises Lactococcus lactis.

The present invention takes advantage of the pili present on the surfaceof many different species of Gram positive bacteria, including Group AStreptococcus (GAS), such as Streptococcus pyogenes (Mora, M., G. et al.(2005) Proc. Natl. Acad. Sci. 102:15641-6.). As used herein, a “pilus”is a hair-like appendage found on the surface of a bacterium.

GAS pili have been shown to mediate attachment to primary humankeratinocytes and to human tonsillar tissue, as well as to severaltissue culture cell lines (Abbot, E. L. et al. (2007) Cell Microbiol.9:1822-1833; Manetti, A. G. et al. (2007). Mol. Microbiol. 64:968-83).They have also been implicated in the formation of biofilms, which maybe important for disease development (Manetti, A. G. et al. (2007) Mol.Microbiol. 64:968-83).

Pili on Gram-positive bacteria are composed of multiple subunits of amajor backbone protein (referred to herein as the pilus shaftpolypeptide) and may also have one or two minor pilin proteins attachedthereto (Mandlik, A. et al. (2008) Trends Microbiol. 16:33-40; Scott, J.R. et al. (2006) Mol. Microbiol. 62:320-30; Telford, J. L. et al. (2006)Nat. Rev. Microbiol. 4:509-19). Gram-positive pilin subunits arecovalently attached to each other and the polymerized pilus iscovalently attached to the peptidoglycan of the cell wall (Swaminathan,A. et al. (2007) Mol. Microbiol. 66:961-974). The minor pilin proteinsare not required for assembly of the pilus, although their presence maybe important for physiological function and specificity of the pili.Prior to the present disclosure, the location of the minor pilins in thepilus structure was unknown and the method by which they are attached tothe shaft was not yet understood.

Pilin proteins have the features typical of Gram-positive surfaceproteins, including an N-terminal signal sequence and a C-terminal cellwall sorting signal (CWSS), which is composed of a hydrophobic domain,beginning with LPXTG (SEQ ID NO: 1) or a similar motif, followed by acharged tail (Schneewind, O. et al. (1993). Embo J. 12:4803-11).

Proteins linked covalently to the Gram-positive cell wall aretranslocated across the cytoplasmic membrane in a Sec-dependent process,which is accompanied by cleavage of the N-terminal signal peptide. Inthe next step, a membrane-associated transpeptidase, referred to as the“housekeeping” sortase, cleaves the CWSS between the threonine (T) andglycine (G) residues of the LPXTG motif, producing an acyl-enzymeintermediate in which the carboxyl group of the threonine of the CWSS islinked to a cysteine residue of the transpeptidase. Subsequently, thethreonine is transferred to an amino group of a constituent of thegrowing cell wall (the peptide crossbridge or diaminopimelic acid),thereby incorporating the protein into the cell wall (for reviews, see,for example, Marraffini et al. (2006) Microbiol Mol Biol Rev 70:192-221and Scott and Barnett (2006) Annu Rev Microbiol 60:397-423), each ofwhich are herein incorporated by reference in its entirety.

The genetic locus in which GAS pili are encoded varies between strainsand has been named the FCT region for the proteins it encodes(Fibronectin-binding, Collagen-binding, T antigen (Bessen, D. E. et al.(2002) Infect. Immun. 70:1159-67). The FCT loci of the GAS strains whosesequence is currently available have been grouped into 6 classes(FCT1-6) based on gene content and gene order (Kratovac, Z. et al.(2007) J. Bacteriol. 189:1299-310, which is herein incorporated byreference in its entirety). A given strain of GAS encodes only a singleFCT locus, and therefore produces only a single type of pilus. GASstrains of the serotypes most common in the western world, M1, M3, M5,M18, and M49, contain either an FCT-2 region (M1) or an FCT-3 region(the others). The genes in these two FCT regions are highly homologousand they occur in the same order in each strain. The presently disclosedmethods and compositions can utilize polypeptides (e.g., pilus tipproteins, pilus shaft polypeptides, tip sortases) from any strain of S.pyogenes bacteria. For example, the polypeptides used in the presentinvention can be a polypeptide encoded by a gene present on a FCT1,FCT2, FCT3, FCT4, FCT5, or FCT6 chromosomal region of a S. pyogenesbacterium. One or more of the polypeptides used in the present inventioncan be a polypeptide encoded by a strain of the serotype M1, M3, M5,M18, or M49 of S. pyogenes bacteria. In particular embodiments, one ormore of the polypeptides are polypeptides encoded by a strain ofserotype M3 of S. pyogenes bacteria. In certain embodiments, one or moreof the polypeptides used in the invention (e.g., pilus shaftpolypeptide, tip sortase, pilus tip polypeptide, pilin chaperonepolypeptide) are encoded by the genes found in the FCT-3 region of theAM3 strain of S. pyogenes.

The protein encoded by the first gene in the FCT-3 operon (FIG. 1), cpa,is a minor pilin protein that has been shown to bind collagen(Podbielski, A. et al. (1999) Mol. Microbiol. 31:1051-64). For the M3strain used in studies presented herein (the AM3 strain), the secondgene (sipA2) is essential for pilus polymerization and probably acts asa chaperone (Zähner, D. et al. (2008) J. Bacteriol. 190:527-35, which isherein incorporated by reference in its entirety). This gene is followedby tee3, which encodes the shaft protein T3, and by srtC2, encoding thepilin polymerase (Barnett, T. C. et al. (2004) J. Bacteriol.186:5865-75, which is herein incorporated by reference in its entirety).Recently Mora et al. elegantly demonstrated that the shaft protein ofGAS pili corresponds to the trypsin-resistant (T) antigen long used forserological typing in GAS (Mora, M. et al. (2005) Proc. Natl. Acad. Sci.102:15641-6). The last gene in the cluster, referred to as orfB , alsoencodes a minor pilin whose homologue was found by immunogold electronmicroscopy to be associated with the pilus structure of a serotype M1strain (Mora, M. et al. (2005) Proc. Natl. Acad. Sci. 102:15641-6). OrfBand Cpa can each be added to the pilus structure in the absence of theother, but, prior to the present disclosure, the residues linking theseminor pilins to the major pilin were not defined.

The pilin proteins in the FCT-2, FCT-3, and FCT-4 regions of GAS strainscontain CWSSs with motifs that differ from the canonical LPXTG (SEQ IDNO: 1) CWSS motif (Barnett, T. C. et al. (2004) J. Bacteriol.186:5865-75). This may indicate that their polymerization requires atranspeptidase different from the housekeeping sortase. This has beendemonstrated for the T3 protein, whose anchoring to the cell wallrequires SrtC2, encoded in the FCT region, and not the housekeeping SrtA(Barnett, T. C. et al. (2004) J. Bacteriol. 186:5865-75). Previousresults indicate that the noncanonical CWSS motif is needed forpolymerization of the T3 protein, as the replacement of thisnoncanonical CWSS motif with the canonical LPSTG (SEQ ID NO: 2) motifprevents formation of T3 polymers (Zähner and Scott (2008) J Bacteriol190:527-535).

The present invention provides Gram-positive bacterium having apolypeptide of interest covalently attached to the tip of a pilusthrough the introduction of a polynucleotide encoding a chimericpolypeptide into the bacterium. The chimeric polypeptide includes thepolypeptide of interest linked in the proper reading frame to a pilustip protein such that the polypeptide of interest is expressed as partof the pili on the transformed bacteria.

The terms “nucleic acid,” “polynucleotide,” or “oligonucleotide”generally are used herein in their art-accepted manners to refer to apolymer of nucleotides. As used herein, an oligonucleotide is typicallyless than 100 nucleotides in length. Polynucleotides can besingle-stranded (with or without a secondary structure, e.g., hairpin)or double-stranded. Naturally occurring nucleic acids includedeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Thepolynucleotide or oligonucleotide may include natural nucleosides (e.g.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine), or syntheticnucleosides, such as, nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,C5-propynylcytidine, C5-propynyluridine, C5-bromouridine,C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine,7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine,and 2-thiocytidine), and/or nucleosides comprising chemically orbiologically modified bases, such as those ribonucleosides that aresubstituted at the 2′ position, for example, with an alkyl or alkyloxygroup (e.g., methylated bases, such as those that are 2′-O-methylated,and 2′-O-methoxyethylated) or a fluoro group, intercalated bases, and/ormodified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose). The phosphate groups in a polynucleotide oroligonucleotide are typically considered to form the internucleosidebackbone of the polymer. In naturally occurring nucleic acids (e.g., DNAor RNA), the backbone linkage is via a 3′ to 5′ phosphodiester bond.Polynucleotides and oligonucletides containing modified backbones ornon-naturally occurring internucleoside linkages, however, also can beused in the presently disclosed subject matter. Such modified backbonesinclude backbones that have a phosphorus atom in the backbone and othersthat do not have a phosphorus atom in the backbone. Examples of modifiedlinkages include, but are not limited to, phosphorothioate and5′-N-phosphoramidite linkages. Polynucleotides and oligonucleotides neednot be uniformly modified along the entire length of the molecule. Forexample, different nucleotide modifications, different backbonestructures, and the like, may exist at various positions in thepolynucleotide or oligonucleotide. Any of the polynucleotides describedherein may utilize these modifications.

According to the presently disclosed methods, a polynucleotidecomprising a nucleotide sequence that encodes a chimeric polypeptide isintroduced into a Gram-positive bacterium. As used herein, the terms“polypeptide” or “peptide” or “protein” can be used interchangeablythroughout, and refer to any monomeric or multimeric protein or peptidecomprised of a polymer of amino acid residues. The term applies to aminoacid polymers in which one or more amino acid residue is an artificialchemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA). A “coding sequence” refersto a nucleotide sequence (e.g., DNA) that encodes a specific RNA orpolypeptide.

The term “expression” has its meaning as understood in the art andrefers to the process of converting genetic information encoded in a DNAsequence (coding sequence) into RNA (e.g., mRNA, rRNA, tRNA, or snRNA)through “transcription” of a polynucleotide (e.g., via the enzymaticaction of an RNA polymerase), and for polypeptide-encodingpolynucleotides, into a polypeptide through “translation” of mRNA. Thus,an “expression product” is, in general, an RNA transcribed from thecoding sequence (e.g., either pre- or post-processing) or a polypeptideencoded by an RNA transcribed from the DNA coding sequence (e.g., eitherpre- or post-modification).

The use of fragments and variants of the disclosed polynucleotides andpolypeptides are also encompassed by the present invention. By“fragment” is intended a portion of the polynucleotide or polypeptideand include active fragments that retain the biological activity of thepolypeptide or the ability to encode an active polypeptide fragment.Alternatively, fragments of a polynucleotide that are useful ashybridization probes or PCR primers need not retain this biologicalactivity. Thus, fragments of a nucleotide sequence may range from atleast about 20 nucleotides, about 50 nucleotides, about 100 nucleotides,about 500 nucleotides, about 1000 nucleotides, and up to the full-lengthpolynucleotide.

Thus, a fragment of the polynucleotide may encode a polypeptide that isbiologically active or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Apolynucleotide that encodes an active polypeptide can be prepared byisolating a portion of the polynucleotide (e.g., by recombinantexpression in vitro) or chemically synthesizing the polynucleotide andassessing the activity of the encoded polypeptide. Polynucleotides thatencode active fragments of the polypeptides of the invention have anucleotide sequence comprising at least 10, 20, 30, 50, 100, 200, 500,or 1000 contiguous nucleotides of the sequences of the invention, or upto the number of nucleotides present in a polynucleotide that encodes afull-length polypeptide.

“Variants” is intended to mean substantially similar sequences. Avariant comprises a polynucleotide having deletions (i.e., truncations)at the 5′ and/or 3′ end; deletion and/or addition of one or morenucleotides at one or more internal sites in the native polynucleotide;and/or substitution of one or more nucleotides at one or more sites inthe native polynucleotide. As used herein, a “native” polynucleotidecomprises a naturally occurring nucleotide sequence. Naturally occurringallelic variants such as these can be identified with the use ofwell-known molecular biology techniques, for example, with polymerasechain reaction (PCR) and hybridization techniques as outlined below.Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis (but which still retain the activity of thepolynucleotides of the invention). Generally, variants of a particularpolynucleotide of the invention will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particularpolynucleotide as determined by sequence alignment programs andparameters as described elsewhere herein.

“Variant” polypeptide is intended to mean a polypeptide derived from thenative polypeptide by deletion (so-called truncation) of one or moreamino acids at the N-terminal and/or C-terminal end of the nativepolypeptide; deletion and/or addition of one or more amino acids at oneor more internal sites in the native polypeptide; or substitution of oneor more amino acids at one or more sites in the native polypeptide.Variant polypeptides encompassed by the present invention arebiologically active, that is they continue to possess the desiredbiological activity of the native polypeptide. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation. Ingeneral, biologically active variants of a native polypeptide of theinvention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the amino acid sequence for the native polypeptideas determined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a polypeptide of theinvention may differ from that polypeptide by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

The polynucleotides that encode the polypeptides useful in thisinvention can be used to isolate variants of the polynucleotidesequences from any organism. In this manner, methods such as PCR,hybridization, and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire polynucleotidessequences set forth herein or to variants and fragments thereof areuseful for the present invention. In a PCR approach, oligonucleotideprimers can be designed for use in PCR reactions to amplifycorresponding polynucleotide sequences from cDNA or genomic DNAextracted from any organism of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See alsoInnis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Methods for preparation of probes for hybridization and for constructionof cDNA and genomic libraries are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

For example, the entire T3 polynucleotide disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding pilus shaft-encoding polynucleotides andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique among pilusshaft-encoding polynucleotide sequences and are optimally at least about10 nucleotides in length, and most optimally at least about 20nucleotides in length. Such probes may be used to amplify correspondingpilus shaft-encoding polynucleotides from a chosen bacterium by PCR.This technique may be used to isolate additional coding sequences from adesired bacterium. Hybridization techniques include hybridizationscreening of plated DNA libraries (either plaques or colonies; see, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions, wherein a probe will hybridize to its target sequence to adetectably greater degree than to other sequences (e.g., at least 2-foldover background). Stringent conditions are sequence-dependent and willbe different in different circumstances. Stringency conditions can beadjusted to allow the identification of 100% complementary sequences orsequences with lower degrees of similarity. Generally, a probe is lessthan about 1000 nucleotides in length, optimally less than 500nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T. can be approximated from theequation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T.=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The T. is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with ≧90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceand its complement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

The percent sequence identity between two sequences can be determinedusing alignment methods that are well known in the art, such asmathematical algorithms. Non-limiting examples of such mathematicalalgorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17;the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math.2:482; the global alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443-453; the search-for-local alignment method of Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm ofKarlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modifiedas in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity,including, but not limited to: CLUSTAL in the PC/Gene program (availablefrom Intelligenetics, Mountain View, Calif.); the ALIGN program (Version2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG WisconsinGenetics Software Package, Version 10 (available from Accelrys Inc.,9685 Scranton Road, San Diego, Calif., USA). Alignments using theseprograms can be performed using the default parameters. The CLUSTALprogram is well described by Higgins et al. (1988) Gene 73:237-244(1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988)Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; andPearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program isbased on the algorithm of Myers and Miller (1988) supra. A PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused with the ALIGN program when comparing amino acid sequences. TheBLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are basedon the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotidesearches can be performed with the BLASTN program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleotidesequence encoding a protein of the invention. BLAST protein searches canbe performed with the BLASTX program, score=50, wordlength=3, to obtainamino acid sequences homologous to a protein or polypeptide of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST2.0) can be used to perform an iterated search that detects distantrelationships between molecules. See Altschul et al. (1997) supra. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTX forproteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10, which uses thealgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, usingthe following parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. The defaultgap creation and extension penalty values can be used for sequencealignments.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The polynucleotides that are introduced into a Gram-positive bacteriumcan further comprise one or more regulatory sequences that are operablylinked to the polynucleotide encoding the chimeric polypeptide thatfacilitate expression of the polynucleotide. “Regulatory sequences”refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. See, forexample, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesmay include promoters, translation leader sequences, introns, andpolyadenylation recognition sequences. A polynucleotide comprisingregulatory sequences operably linked to coding sequences can be referredto as expression cassettes.

Regulatory sequences are operably linked with a coding sequence to allowfor expression of the polypeptide encoded by the coding sequence.“Operably linked” is intended to mean a functional linkage between twoor more elements. For example, an operable linkage between apolynucleotide that encodes a polypeptide and a regulatory sequence(i.e., a promoter) is a functional link that allows for expression ofthe polypeptide. Operably linked elements may be contiguous ornon-contiguous. Polynucleotides may be operably linked to regulatorysequences in sense or antisense orientation. When used to refer to thejoining of two protein coding regions, by operably linked is intendedthat the coding regions are in the same reading frame.

The regulatory regions (i.e., promoters, transcriptional regulatoryregions, and translational termination regions) and/or the codingpolynucleotides may be native/analogous to the host cell or to eachother. Alternatively, the regulatory regions and/or the codingpolynucleotides may be heterologous to the host cell or to each other.As used herein, “heterologous” in reference to a sequence or apolypeptide is a sequence or polypeptide that originates from a foreignspecies, or, if from the same species, is substantially modified fromits native form in composition and/or genomic locus by deliberate humanintervention. For example, a promoter operably linked to a heterologouspolynucleotide is from a species different from the species from whichthe polynucleotide was derived, or, if from the same/analogous species,one or both are substantially modified from their original form and/orgenomic locus, or the promoter is not the native promoter for theoperably linked polynucleotide.

In particular embodiments wherein the polynucleotide encoding thechimeric polypeptide comprises regulatory sequences, the polynucleotidecan further comprise additional coding sequences. In some of theseembodiments, the regulatory sequences can be operably linked to morethan one coding sequence. For example, a single promoter can be operablylinked to more than one coding sequence, wherein the coding sequencesare co-transcribed from the single promoter into a single polycistronictranscript, which is separately translated into more than onepolypeptide.

It will be appreciated by those skilled in the art that the design ofthe expression cassette can depend on such factors as the choice of thehost cell to be transformed, the level of expression of the presentlydisclosed polynucleotides, and the like. Such expression cassettestypically include one or more appropriately positioned sites forrestriction enzymes, to facilitate introduction of the nucleic acid intoa vector.

It will further be appreciated that appropriate promoter and/orregulatory elements can readily be selected to allow expression of thepresently disclosed polynucleotides in the cell of interest.

“Promoter” refers to a polynucleotide capable of controlling theexpression of a polynucleotide. In general, the polynucleotide to betranscribed is located 3′ to a promoter sequence. The promoter sequencemay comprise proximal and more distal upstream elements; the latterelements often referred to as enhancers. Accordingly, an “enhancer” is apolynucleotide, which can stimulate promoter activity, and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is recognized that sincein most cases the exact boundaries of regulatory sequences have not beencompletely defined, polynucleotide fragments of different lengths mayhave identical promoter activity.

The promoters used in accordance with the present invention may beconstitutive promoters or regulated promoters. Common examples of usefulregulated promoters include those of the family derived from the nisinpromoter (see, for example, U.S. Pat. No. 5,914,248 and Kleerebezem etal. (1997) Appl Environ Microbiol 63:4581-4584, each of which are hereinincorporated by reference in its entirety); and thetetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, which are herein incorporated byreference). Regulated promoters utilize promoter regulatory proteins inorder to control transcription of the gene of which the promoter is apart. Where a regulated promoter is used herein, corresponding promoterregulatory protein(s) will also be part of an expression systemaccording to the present invention. Examples of promoter regulatoryproteins include the NisR and NisK proteins for use with the nisApromoter. Many regulated-promoter/promoter-regulatory protein pairs areknown in the art.

Promoter regulatory proteins interact with or are activated or repressedby an effector compound, i.e. a compound that reversibly or irreversiblyassociates with or activates or represses the regulatory protein so asto enable the protein to either release or bind to at least one DNAtranscription regulatory region of the gene that is under the control ofthe promoter, thereby permitting or blocking the action of atranscriptase enzyme in initiating transcription of the gene. Anon-limiting example of an effector compound is tetracycline or nisinfor use with tetracycline-regulated promoter systems or nisin-regulatedsystems, respectively. Effector compounds are classified as eitherinducers or co-repressors, and these compounds include native effectorcompounds and gratuitous inducer compounds. Manyregulated-promoter/promoter-regulatory-protein/effector-compound systemsare known in the art. Although an effector compound can be usedthroughout the cell culture or fermentation, in some embodiments inwhich a regulated promoter is used, after growth of a desired quantityor density of host cell biomass, an appropriate effector compound isadded to the culture to directly or indirectly result in expression ofthe desired gene(s) encoding the protein or polypeptide of interest.

Other non-limiting examples of useful promoters for expression in Grampositive cells are the P_(ami), P_(spac), P_(veg), and P23 promoters(see, for example Biswas et al. (2008) Microbiology 154:2275-2282, whichis herein incorporated by reference in its entirety).

Other regulatory elements may be included in an expression cassette,including but not limited to, transcriptional enhancer sequences,translational enhancer sequenes, other promoters, activators,translational start and stop signals, transcription terminators,cistronic regulators, polycistronic regulators, signal sequences (e.g.,Sec dependent signal sequences), or tag sequences, such as nucleotidesequence “tags” and “tag” polypeptide coding sequences, whichfacilitates identification of the polypeptide or cell expressing thepolypeptide. A non-limiting example of a tag polypeptide is thehemagglutinin (HA) peptide.

Regulatory sequences found within expression cassettes can include a 3′non-coding region. The “3′ non-coding region” or “terminator region”refers to DNA or RNA sequences located downstream of a coding sequenceand may include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by effecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

Proteins linked covalently to the Gram-positive cell wall aretranslocated across the membrane in a Sec-dependent process accompaniedby cleavage of the signal peptide by a signal peptidase. Thus, in someembodiments, the expression cassette comprises a nucleotide sequencethat encodes for an appropriate signal peptide that is inserted into theexpression cassette in such a manner that it encodes a polypeptide ofinterest with the signal peptide fused to the amino terminal end of thepolypeptide of interest. Sec-dependent signal sequences are known in theart and generally consist of a short (about 30 amino acids), mainlyhydrophobic sequence comprising the following three domains: (i) apositively charged n-region with at least one arginine or lysineresidue, (ii) a hydrophobic h-region and (iii) an uncharged but polarc-region. The cleavage site for the signal peptidase is located in thec-region. However, the degree of signal sequence conservation andlength, as well as the cleavage site position, can vary betweendifferent proteins. The signal sequence aids protein export and iscleaved off by a periplasmic signal peptidase when the exported proteinreaches the periplasm. In some embodiments, the signal peptide encodedby the expression cassette comprises the signal peptide derived from theS. pyogenes pilus tip polypeptide that is fused to the polypeptide ofinterest to be displayed on the pili tip (such as the signal peptide setforth in SEQ ID NO: 100 from the Cpa protein). In other embodiments, thesignal peptide is derived from the polypeptide of interest. The signalpeptide can also be heterologous to both the polypeptide of interest andthe S. pyogenes pilus tip polypeptide (such as a consensus Sec-dependensignal sequence).

For suitable expression systems for prokaryotic cells, see Chapters 16and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), whichare herein incorporated by reference. See also Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.), which is herein incorporated by reference in itsentirety.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT).Additional selectable markers include phenotypic markers such asβ-galactosidase and fluorescent proteins such as green fluorescentprotein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter etal. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolteet al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) PlantPhysiol 129:913-42), and yellow florescent protein (for example, PhiYFP™from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). Foradditional selectable markers, see generally, Yarranton (1992) Curr.Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad.Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al., (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); andGill et al. (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting asany selectable marker gene can be used in the present invention.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Such expression cassettes can be contained in a vector which allow forthe introduction of the expression cassette into a cell. In specificembodiments, the vector allows for autonomous replication of theexpression cassette in a cell or may be integrated into the genome of acell. Such vectors are replicated along with the host genome. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids (vectors). However, the invention isintended to include such other forms of expression vectors, such asviral vectors.

According to the present invention, polynucleotides encoding thechimeric polypeptides are introduced into a cell. “Introducing” isintended to mean presenting to the cell the polynucleotide in such amanner that the sequence gains access to the interior of the cell. Themethods of the invention do not depend on a particular method forintroducing a sequence into a cell, only that the polynucleotide gainsaccess to the interior of the cell. Methods for introducingpolynucleotides into cells are known in the art including, but notlimited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

“Stable transformation” is intended to mean that the polynucleotideintroduced into a cell integrates into the genome of the cell and iscapable of being inherited by the progeny thereof “Transienttransformation” is intended to mean that a polynucleotide is introducedinto the cell and does not integrate into the genome of the cell.

Exemplary art-recognized techniques for introducing foreignpolynucleotides into a host cell include calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, particle gun, or electroporation and viral vectors.Suitable methods for transforming or transfecting host cells can befound in U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat.No. 4,897,355, Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.)and other standard molecular biology laboratory manuals. One of skillwill recognize that depending on the method by which a polynucleotide isintroduced into a cell, the polynucleotide can be stably incorporatedinto the genome of the cell, replicated on an autonomous vector orplasmid, or present transiently in the cell. In some embodiments,transient expression may be desired. In those cases, standard transienttransformation techniques may be used. Such methods include, but are notlimited to viral transformation methods, and microinjection of DNA orRNA, as well other methods well known in the art.

Host organisms containing the introduced polynucleotide are referred toas “transgenic” or “transformed” organisms. By “host cell” is meant acell that contains an introduced polynucleotide construct and supportsthe replication and/or expression of the construct. The host cells ofthe present invention are Gram-positive bacteria.

The skilled artisan will recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al.(1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events mayhave to be screened in order to obtain cells displaying the desiredexpression level and pattern. Such screening may be accomplished by PCRor Southern analysis of DNA to determine if the introducedpolynucleotide is present in complete form, and then northern analysisor RT-PCR to determine if the expected RNA is indeed expressed.

According to the present invention, a polypeptide of interest isdisplayed on the surface of a bacterium as a chimeric polypeptide,wherein the polypeptide of interest is covalently attached directly orindirectly to a pilus tip protein. As used herein, a “chimericpolypeptide” or “fusion polypeptide” refers to a polypeptide comprisingtwo polypeptides operably linked to one another, wherein the twopolypeptides are not covalently bound to one another through peptidebonds in nature (without any human intervention). As described elsewhereherein, “operably linked” is intended to mean a functional linkagebetween two or more elements. For example, two polypeptides within achimeric polypeptide are operably linked if the two polypeptides arefused to each other directly or indirectly through a peptide bond sothat both polypeptides fulfill the proposed function attributed to eachpolypeptide. The chimeric polypeptides of the invention are createdthrough the joining of the coding sequences for each polypeptide,wherein the two coding sequences are operably linked within the samereading frame to allow for the expression of the chimeric polypeptide.The polypeptide of interest could be fused indirectly to the pilus tipprotein or active fragment or variant thereof, wherein additional aminoacid residues can serve as a linker between the two polypeptides. Theuse of a linker sequence can increase the likelihood that the twopolypeptides (polypeptide of interest and pilus tip protein) foldproperly. The linker sequence can consist of 1 amino acid to about 100amino acid residues or more.

The chimeric polypeptides of the invention comprise a heterologouspolypeptide and a Gram-positive bacterial pilus tip polypeptide (or anactive variant or fragment thereof). As used herein, a “pilus tippolypeptide” or “pilus tip protein” is a polypeptide that is present atthe end of a bacterial pilus that extends out from the surface of thebacteria. The pilus tip polypeptide is distinct from the major pilinpolypeptide that forms the shaft of the pilus (the pilus shaftpolypeptide). While pilus shaft polypeptides can be localized at the tipof some pili, they are not considered pilus tip polypeptides, as theyalso comprise the major proteins found within the pilus shaft. Ingeneral, the pilus shaft polypeptide is the major pilin polypeptide andthe pilus tip polypeptide is a minor pilin polypeptide within a givenGram-positive bacterium. In general, the major pilin protein is the Tantigen that is often used for serological typing of S. pyogenes (Moraet al. (2005) Proc Natl Acad Sci USA 102:15641-15646; Schneewind et al.(1990) J Bacteriol 172:3310-3317).

In some embodiments, the pilus tip polypeptide comprises a pilus tippolypeptide from a Streptococcus bacterium. In some of theseembodiments, the pilus tip polypeptide comprises a Streptococcuspyogenes pilus tip polypeptide. Data presented elsewhere hereindemonstrate that the minor pilin protein Cpa from the M3 strain ofStreptococcus pyogenes is present on the tip of pili. Cpa is a putativeadhesin protein that is capable of binding to collagen. Thus, in someembodiments, the pilus tip polypeptide is an adhesin. An “adhesin” is apolypeptide that binds to an extracellular matrix protein, host cellsurface protein, or other host cell-associated protein that facilitatesbacterial-host cell interactions. An additional, non-limiting example ofa Streptococcus pyogenes adhesin protein is the fibronectin-bindingprotein F1.

In general, the pilus tip protein of any given Streptococcus pyogenesstrain is the protein encoded by the first non-regulatory gene presentin the FTC region of the bacterial chromosome. In some embodiments, theS. pyogenes pilus tip polypeptide can be selected from the groupconsisting of Cpa, protein F1, OrfB, Spy0128, Spy0130, FctA, FctX, andFctB. In particular embodiments, the pilus tip polypeptide comprises aCpa polypeptide (also known as Cpa49). In some of these embodiments, thepilus tip polypeptide comprises the Cpa polypeptide from the AM3 strainof S. pyogenes (sequence set forth in SEQ ID NO: 3), which is encoded bythe nucleotide sequence set forth in SEQ ID NO: 4.

To determine if a given polypeptide functions as a pilus tip protein,one can use assays that are known in the art to localize a polypeptideto the tip of a pilus, including but not limited to assays presentedelsewhere herein (see Experimental Example 1). Assays used to determineif a polypeptide is polymerized into a pilus structure, in general,involve extracting the cell wall fraction of bacteria (with mutanolysinwith or without lysozyme), boiling the extract in SDS and separating theproteins using SDS-PAGE. Pilus proteins appear as high molecular weightladders in immunoblots. The E. coli expression system and the mutationalanalysis of the pilus shaft polypeptide and pilus tip polypeptide usedelsewhere herein can be used to determine if the polypeptide is indeedlocalized to the pilus tip. Other methods known in the art can be usedto localize the pilus tip protein to the leading edge of pili, includingbut not limited to, visualization by fluorescence microscopy or negativestaining (e.g., immunogold electron microscopy).

The chimeric polypeptide displayed on the tip of the Gram-positive pilican comprise an active variant or fragment of a pilus tip polypeptide.An active variant or fragment of a pilus tip polypeptide is apolypeptide that retains the ability to be localized to the tip of abacterial pilus. In some embodiments, the active variant or fragment ofthe pilus tip polypeptide comprises the cell wall sorting signal (CWSS).In particular embodiments, the active fragment of the pilus tippolypeptide comprises at least one amino acid residue amino terminal to(i.e., preceding) the CWSS and the CWSS itself. In certain embodiments,the polypeptide fragment of the S. pyogenes pilus tip polypeptidecomprises at least 2, at least 3, at least 5, at least 10, at least 20,at least 30, at least 40, at least 50, at least 100, at least 200, atleast 300, at least 400, at least 500, at least 1000 amino acid residuesamino terminal to the CWSS up to the full length pilus tip polypeptidesequence. In some embodiments, an active fragment comprises amino acids594-744 of SEQ ID NO: 3 (this region is set forth in SEQ ID NO: 6). Insome embodiments, the polypeptide of interest is fused (directly orindirectly) to an amino acid sequence that has at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or higher sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 6.

In some embodiments, active variants of the pilus tip polypeptide havean amino acid sequence having at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or higher sequence identity to the amino acidsequence set forth in SEQ ID NO: 3. In certain embodiments, activevariants of the pilus tip polypeptide are encoded by a nucleotidesequence having at least 50%, at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or higher sequence identity to the nucleotide sequence setforth in SEQ ID NO: 4.

The cell wall sorting signal is present in all Gram-positive bacterialsurface displayed proteins and is comprised of the cell wall sortingsignal (CWSS) motif, which is generally a five amino acid residue motif,a hydrophobic domain carboxyl to the CWSS motif, and a charged tailregion carboxyl to the substantially hydrophobic domain. The CWSS motifis recognized and cleaved by the “housekeeping” sortase A, which is amembrane-associated transpeptidase. Canonical CWSS motifs generallycomprise a LPXTG (SEQ ID NO: 1) amino acid sequence. The motif isgenerally cleaved at the threonine by the sortase to form an acyl-enzymeintermediate, wherein the carboxyl group of the threonine (T) of theCWSS is linked to a cysteine (C) residue of the transpeptidase.Subsequently, the threonine is transferred to an amino group of thepeptidoglycan molecule within the peptide crossbridge of the growingcell wall, thereby incorporating the protein into the cell wall (forreviews see Marraffini, L. A. et al. (2006) Microbiol. Mol. Biol. Rev.70:192-221; Scott, J. R. et al. (2006) Annu. Rev. Microbiol.60:397-423). As used herein, a “cleaved CWSS motif” comprises theremains of a CWSS motif sequence following the cleavage of the motif bya sortase transpeptidase enzyme. A cleaved canonical CWSS motif, thus,has the sequence of LPXT.

The S. pyogenes pilus tip protein or variant or fragment thereof that iscarboxy terminal to a polypeptide of interest displayed on the tip of apilus on a Gram-positive bacterium comprises a cleaved cell wall sortingsignal (CWSS) motif. In some embodiments, variants of the pilus tipprotein have an amino acid sequence having at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or higher sequence identity tothe amino acid sequence set forth in SEQ ID NO: 101. In otherembodiments, the variant of the pilus tip protein comprising a cleavedCWSS motif has an amino acid sequence having at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or higher sequence identity tothe amino acid sequence set forth in SEQ ID NO: 103. In certainembodiments, the pilus tip protein comprising a cleaved cell wallsorting signal motif has the sequence set forth in SEQ ID NO: 101. Inother embodiments, the pilus tip protein comprising a cleaved cell wallsorting signal motif has the sequence set forth in SEQ ID NO: 103.

Polymerization of pilin proteins in Gram-positive bacteria requires asortase family transpeptidase (pilin polymerase) and therefore isgenerally assumed to proceed by a process similar to that demonstratedfor the Staphylococcus aureus housekeeping sortase (Ton-That, H. et al.(1999) Proc. Natl. Acad. Sci. 96:12424-9; Ton-That, H. (2004) TrendsMicrobiol. 12:228-34; for a review of sortases, see Marrafifini et al.(2006) Microbiol Mol Biol Rev 70:192-221, both of which are hereinincorporated in their entireties). It is believed that the pilinpolymerase catalyzes formation of a peptide bond between the threoninein the CWSS motif of one subunit and an ε-amino group of a lysine in thenext subunit of the growing pilus chain.

Along with the five amino acid residue CWSS motif, the CWSS alsocomprises a carboxyl terminal substantially hydrophobic domain and acharged tail region. By “substantially hydrophobic” is intended a regionof a polypeptide, wherein at least about 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or more of the amino acid residuesmaking up the region are hydrophobic. In some embodiments, thehydrophobic region is at least about 20, at least 25, at least 30, atleast 35, at least 40, at least 45, at least 50, at least 55, or atleast 60 amino acid residues in length. In particular embodiments, thehydrophobic region is at least about 25 amino acid residues in length.In certain embodiments, the hydrophobic region comprises the sequenceset forth in amino acids 714-738 of SEQ ID NO: 3.

The hydrophobic region of the CWSS is followed by a charged tail region.At least about 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or more of the amino acid residues making up the charged tailregion have a positive or negative charge at physiological pH. In someembodiments, the charged tail region comprises about 5 to about 20 aminoacid residues, including, but not limited to, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, and 20. In certain embodiments, the chargedtail region comprises the sequence RKGTKK (SEQ ID NO: 5), whichcorresponds to the extreme carboxy terminus of Cpa (set forth in SEQ IDNO: 3).

It was demonstrated elsewhere herein that the sortase C2 polypeptide iscapable of polymerizing the major pilin T3 polypeptide and is requiredfor the covalent attachment of the minor pilin Cpa to T3 polypeptides atthe tip of pili. Thus, according to the presently disclosed methods andcompositions, the Gram-positive bacteria useful for the display ofpolypeptides of interest express a tip sortase as well as a pilus shaftpolypeptide. As used herein, a “tip sortase” is a sortase enzyme capableof covalently attaching a pilus tip polypeptide to the pilus shaft. Thetip sortase can be from any organism. In some embodiments, a tip sortasecomprises a sortase C enzyme. In particular embodiments, the tip sortasecomprises a sortase C1 or sortase C2 enzyme. In some embodiments, thesortase C enzyme comprises a SrtC1 polypeptide, which is found in the M1strains of S. pyogenes (see Barnett et al. (2004) J Bacteriol186:5865-5875).

In other embodiments, the tip sortase comprises a sortase C2 enzyme,such as the sortase C2 enzyme encoded within the FCT-3 and FCT-4chromosomal regions of S. pyogenes bacteria (including, but not limitedto the FCT-3 or FCT-4 regions from M3, M5, M12, M18, and M49 strains ofS. pyogenes; see Barnett et al. (2004) J Bacteriol 186:5865-5875).

In particular embodiments, the sortase C2 enzyme comprises the srtC2from the AM3 strain of S. pyogenes (with the amino acid sequence setforth in SEQ ID NO: 7). In some embodiments, the tip sortase has anamino acid sequence having at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 96%, atleast 97%, at least 98%, at least 99%, or more sequence identity to thesequence set forth in SEQ ID NO: 7. The AM3 sortase C2 polypeptide isencoded by the nucleotide sequence set forth in SEQ ID NO: 8. In someembodiments, the tip sortase is encoded by a nucleotide sequence havingat least about 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 96%, at least 97%, at least98%, at least 99%, or more sequence identity to the sequence set forthin SEQ ID NO: 8. Active variants of SEQ ID NO: 7 retain the ability toattach a pilus tip polypeptide to a pilus shaft. Suitable assays fordetermining if a given polypeptide exhibits this activity include anymethod known in the art or described elsewhere herein (see ExperimentalExample 1).

Previous results have demonstrated that the sortase C2 enzyme is capableof attaching the T3 shaft polypeptide of the pilus from the M3 strain ofS. pyogenes to the cell wall through a non-canonical CWSS motif. TheCWSS motif of the T3 polypeptide (also referred to as Orf100) comprisesa QVPTG (set forth in SEQ ID NO: 9) amino acid sequence. Resultspresented elsewhere herein demonstrate this non-canonical CWSS motif isalso utilized by SrtC2 to attach the T3 polypeptides to one another.Further presented herein are data that show the SrtC2 enzyme alsocatalyzes the covalent attachment of the Cpa minor pilin to the T3protein. Similar to the T3 polypeptide, Cpa also comprises anon-canonical CWSS motif with the amino acid sequence of VPPTG (SEQ IDNO: 10). These data suggest sortase C2 polypeptides recognize and cleavenon-canonical CWSS motifs. Thus, in some embodiments, both the majorshaft polypeptide expressed by the Gram-positive bacterium and the pilustip polypeptide (or active variant or fragment thereof) fused to thedisplayed heterologous polypeptide comprise a non-canonical CWSS motif.In particular embodiments, a tip sortase is one that is capable ofcovalently attaching a pilus tip polypeptide with a non-canonical CWSSmotif to a growing pilin chain or polymerizing a pilus shaft polypeptidehaving a non-canonical CWSS motif. As used herein, a “non-canonical CWSSmotif” is one wherein the sequence does not follow the consensuscanonical CWSS motif of LPXTG (SEQ ID NO: 1), wherein X is any aminoacid. In some embodiments, the non-canonical CWSS motif comprises aXXPTG (SEQ ID NO: 11) motif. In some of these embodiments, the firstamino acid comprises a glutamine or a valine. In other embodiments, thesecond amino acid comprises a valine or a proline. In yet otherembodiments, the first amino acid comprises a glutamine or a valine andthe second amino acid comprises a valine or a proline. In certainembodiments, the non-canonical motif is one comprising a XXPTG motif(SEQ ID NO: 11), wherein the first amino acid is not a leucine residue.In other embodiments, the second amino acid residue is not a proline.

The Gram-positive bacteria of the invention comprise a major pilin thatfunctions as the pilus shaft polypeptide. A “pilus shaft polypeptide” isa polypeptide that comprises the shaft of the pilus. In someembodiments, the pilus shaft polypeptide comprises at least about 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%,96%, 97%, 98%, 99%, or higher of the polypeptides comprising a pilus. Insome embodiments, the pilus shaft polypeptide comprises the major pilinprotein. In general, the major pilin protein is the T antigen that isoften used for serological typing of S. pyogenes (Mora et al. (2005)Proc Natl Acad Sci USA 102:15641-15646; Schneewind et al. (1990) JBacteriol 172:3310-3317). In some embodiments, the pilus shaftpolypeptide comprises a non-canonical CWSS motif within its cell wallsorting signal. In particular embodiments, the pilus shaft polypeptidecomprises the T3 polypeptide. In some of these embodiments, the T3polypeptide is from a M3 strain of S. pyogenes. In some of theseembodiments, the T3 polypeptide comprises the T3 polypeptide from theAM3 strain of S. pyogenes, the amino acid sequence of which is set forthin SEQ ID NO: 12. In particular embodiments, the pilus shaft polypeptidehas an amino acid sequence having at least about 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or high sequence identity to thesequence set forth in SEQ ID NO: 12. The AM3 T3 polypeptide is encodedby the nucleotide sequence set forth in SEQ ID NO: 13. In someembodiments, the pilus shaft polypeptide is encoded by a nucleotidesequence having at least about 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher sequence identity to the nucleotide sequenceset forth in SEQ ID NO: 13. Pilus shaft polylpeptides and activevariants thereof retain the ability to polymerize into a pilus and to becovalently attached to a peptidoglycan molecule in the bacterial cellwall. Assays for detecting this activity include any method known in theart (see, for example, Barnett et al. (2004) J Bacteriol 186:5865-5875)and methods disclosed elsewhere herein (see Experimental Example 1). Asdisclosed elsewhere herein, the lysine residue at position 173 withinthe T3 protein is required for covalent attachment of T3 to Cpa.Specifically, the K173 residue is covalently attached to the threonineresidue within the CWSS motif of Cpa. The lysine corresponding to aminoacid residue 173 of T3 is conserved throughout the major pilin proteinsfound in at least the FCT-2, FCT-3, and FCT-4 chromosomal regions (seeFIG. 5). Thus, in some embodiments, the pilus shaft polypeptidecomprises a lysine residue within the major pilin protein in a similarregion of the polypeptide as the K173 in T3 protein, which can bedetermined through alignment of the sequences using methods describedelsewhere herein.

In some embodiments of the present invention, the tip sortase functionsas a pilin polymerase, facilitating the polymerization of the pilinshaft polypeptides, in addition to its role in attaching the pilus tippolypeptide to the pilin shaft polypeptide. Additionally, in certainembodiments, the tip sortase has the ability to attach the pilus topeptidoglycans within the cell wall. In other embodiments, the tipsortase that attaches the pilus tip polypeptide to the pilus shaftpolypeptide is distinct from the pilin polymerase that facilitatesattachment of the pilus shaft polypeptides to one another or is distinctfrom the sortase enzyme that attaches the pilus to the cell wall (e.g.,the housekeeping sortase A). In these embodiments, the transformedGram-positive bacteria further express or comprise a pilin polymerasethat can polymerize the pilin shaft polypeptide, a sortase that attachesthe pilus to the cell wall (e.g., the housekeeping sortase A), or both.

Attachment of the T3 polypeptide and the Cpa polypeptide to the cellwall of M3 strains of S. pyogenes requires the SipA pilin chaperonepolypeptide. As used herein, a pilin chaperone polypeptide is apolypeptide that is required for the stabilization of pilin proteins andthat facilitates the polymerization and cell wall attachment of a pilus.Thus, in some embodiments, the Gram-positive bacteria further express apilin chaperone polypeptide. In certain embodiments, the pilin chaperonepolypeptide comprises a SipA polypeptide from an M3 strain of S.pyogenes (Zähner and Scott (2008) J Bacteriol 190:527-535). In someembodiments, the SipA polypeptide is from the AM3 strain of S. pyogenes,the amino acid sequence of which is set forth in SEQ ID NO: 14. In someof these embodiments, the pilin chaperone polypeptide has an amino acidsequence having at least about 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher sequence identity to the sequence set forthin SEQ ID NO: 14. The AM3 SipA polypeptide is encoded by the nucleotidesequence set forth in SEQ ID NO: 15. In some embodiments, the pilinchaperone polypeptide is encoded by a nucleotide sequence having atleast about 50%, at least 60%, at least 70%, at least 80%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99% orhigher sequence identity to the sequence set forth in SEQ ID NO: 15. Agiven polypeptide sequence (or a polypeptide encoded by any nucleotidesequence) can be assessed for its ability to function as a pilinchaperone using any method known in the art (see Zähner and Scott (2008)J Bacteriol 190:527-535), including those methods disclosed elsewhereherein (see Experimental Example 1).

In particular embodiments wherein Cpa or an active variant or fragmentthereof serves as the pilus tip protein that is fused to the polypeptideof interest, the Gram positive bacterium further expresses or comprisesthe tip polymerase SrtC2, the T3 pilus shaft polypeptide, and the SipA2chaperone protein. In some of these embodiments, the SrtC2 tippolymerase has the sequence set forth in SEQ ID NO: 7, the T3 pilusshaft polypeptide has the sequence set forth in SEQ ID NO: 12, and theSipA2 chaperone protein has the sequence set forth in SEQ ID NO: 14.

In some embodiments, at least one of the pilus shaft polypeptide,sortase C polypeptide, and pilin chaperone polypeptide are heterologousto the Gram-positive bacterium that is displaying a polypeptide ofinterest. In some embodiments, one or all of the polypeptides can beexpressed within the bacteria through the introduction of an expressioncassette that comprises a polynucleotide that comprises a nucleotidesequence that encodes for at least one of the three polypeptides. Insome embodiments, the expression cassette that comprises thepolynucleotide that encodes the chimeric polypeptide also comprises acoding sequence for at least one of a SrtC, pilus shaft, and SipApolypeptides. In other embodiments, the expression cassette thatcomprises a polynucleotide sequence that encodes at least one of theSrtC, pilus shaft, and SipA polypeptides is different from theexpression cassette that comprises the polynucleotide that encodes thechimeric polypeptide. In yet other embodiments, each of the polypeptidesis encoded by a coding sequence present on a distinct expressioncassette.

The transformed Gram-positive bacterium displaying the polypeptide ofinterest can display more than one polypeptide of interest. In some ofthese embodiments, the Gram-positive bacterium comprises at least twogroups of pili, wherein each group expresses a distinct polypeptide ofinterest. This can be due to the introduction of at least two distinctpolynucleotides, each encoding for a distinct chimeric polypeptide.Alternatively, one polynucleotide can be introduced into the bacterium,wherein the polynucleotide comprises coding sequences for each of thepolypeptides that are to be displayed on the surface of the bacterium.In these embodiments, the coding sequence for each chimeric polypeptide(the polypeptide of interest fused to a Gram positive bacterial pilustip protein or an active variant or fragment thereof) can be operablylinked to the same regulatory sequences (monocistronic) or to separateregulatory sequences (polycistronic).

According to the methods of the invention, following the introduction ofthe polynucleotide comprising an expression cassette encoding thechimeric polypeptide, the Gram-positive bacterium is grown underconditions that allow for the generation of the pilus. The growthconditions used for this step of the presently disclosed methods can beany growth condition known in the art for growth of the Gram-positivebacterium that is displaying the polypeptide of interest. In general,the bacteria can be grown in liquid or solid culture medium. Growth inliquid culture often is facilitated through aeration of the culturemedium (e.g., through shaking of the container comprising the medium).In some embodiments, particularly those embodiments wherein theGram-positive bacterium is a S. pyogenes bacterium, the bacterium isgrown in Todd-Hewitt medium (such as the Todd-Hewitt medium that iscommercially available from BD, Sparks, Md.). In some of theseembodiments, growth supplements are added to the medium. A non-limitingexample of a growth supplement is yeast extract. In some embodiments,Todd-Hewitt medium is supplemented with yeast extract at a 0.2%concentration. Another non-limiting example of a growth media for Grampositive bacteria, including L. lactis, is M17 media (such as the M17media available from Oxoid Limited, Hampshire, UK). The M17 media can besupplemented with glucose (for example, at a concentration of 0.5%).

As used herein, a “polypeptide of interest” refers to any full-length,variant, or fragment of any naturally-occurring polypeptide from anyorganism (prokaryotic or eukaryotic) or a synthetically derivedpolypeptide that would find use in the display on the surface of abacterium. In some embodiments, the polypeptide of interest that isdisplayed on the surface of the bacterium retains the activity (e.g.,enzymatic activity) of the naturally occurring polypeptide or the samepolypeptide that has not been fused to the pilus tip polypeptide. Asnon-limiting examples, the polypeptide can comprise an enzyme, anantigen, or a biosorbent. The polypeptide of interest may be native tothe Gram-positive bacterium that is displaying the polypeptide ofinterest or the polypeptide of interest may be heterologous to thebacterium.

The compositions and methods of the invention can be used for any useknown in the art for surface displayed polypeptides (see, for example,Hansson et al. (2001) Combinatorial Chemistry & High ThroughputScreening 4:171-184; Wu et al. (2008) Trends in Microbiology 16:181-188;Wernerus and Stahl (2004) Biotechnol. Appl. Biochem. 40:209-228; Chenand Georgiou (2002) Biotechnol Bioeng 79:496-503; Lee et al. (2003)Trends in Biotechnology 21:45-52; Wernerus et al. (2002) Journal ofBiotechnology 96:67-78, each of which are herein incorporated byreference). For example, the compositions and methods of the inventionare useful in methods for inducing an immunological response in asubject, methods for screening for expression of a heterologouspolypeptide, methods for removing a contaminant from a composition(e.g., soil, water), methods for producing ethanol, and methods forimproving food and nutritional additives.

As used herein, an “antigen” comprises any polypeptide that can mount animmune response in a subject and is, thus, immunologically active. Thepresent invention provides immunological compositions or vaccinescomprising a Gram-positive bacterium displaying an antigen on the tip ofa pilus, wherein the antigen is amino terminal to a Streptococcuspyogenes pilus tip protein or an active variant or fragment thereof,wherein said pilus tip protein or active variant or fragment thereofcomprises a cleaved cell wall sorting signal (CWSS) motif.

The immunological compositions comprising the Gram-positive bacteriadisplaying an antigen can be used to prevent the development of aparticular disease or unwanted condition through the administration ofthe compositions to a subject. As used herein, the term “prevent” refersto obtaining a desired pharmacologic and/or physiologic effect.Administration of the immunological composition might lead to completeor partial prevention of a particular infection or disease or sign orsymptom thereof.

Methods for inducing an immunological response in a subject compriseadministering to a subject a composition comprising a Gram-positivebacterium displaying an antigen on the tip of a pilus, wherein theantigen is amino terminal to a Streptococcus pyogenes pilus tip proteinor an active variant or fragment thereof, wherein said active variant orfragment comprises a cleaved cell wall sorting signal (CWSS) motif.

When referring to the Gram-positive bacteria of the invention orcompositions comprising the same, the term “administering,” andderivations thereof, comprises any method that allows for theGram-positive bacteria or compositions comprising the same to contact acell within the subject to which the composition was administered.

By “subject” is intended an animal, including a mammal, such as a human,and including, but by no means limited to, domestic animals, such asfeline or canine subjects, farm animals, such as but not limited tobovine, equine, caprine, ovine, and porcine subjects, wild animals(whether in the wild or in a zoological garden), research animals, suchas mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avianspecies, such as chickens, turkeys, songbirds, etc., i.e., forveterinary medical use. In particular embodiments, the subject is ahuman.

A non-limiting example of an antigen that can be displayed on the tip ofa Gram-positive bacterial pili includes domain 1′ (residues 168-258) ofthe protective antigen of the anthrax toxin of Bacillus anthracis (thenucleotide and amino acid sequence of which is set forth in SEQ ID NO 91and 92, respectively), which is the domain that remains at theN-terminus of the toxin following its proteolytic cleavage by proteasesubiquitously present in host tissue. This domain, called the “LEFdomain”, is involved in binding to the other subunits of the anthraxtoxin, LF (lethal factor), and EF (edematous factor). This antigen isuseful for methods involving the administration of the Gram-positivebacterium displaying such an antigen for the purposes of providingprotection against an infection of Bacillus anthracis. Another domain ofthe anthrax toxin that can be used as an antigen for the purpose ofstimulating an immunological response to a Bacillus anthracis bacteriais domain 4 (residues 596-735 of SEQ ID NO: 92), called “RBD”, which isresponsible for binding of the toxin to host cell receptors. The RBD andLEF domains are antigenic as DNA vaccines, have been inserted into theinfluenza virus fused within the hemagglutinin protein, and have beenshown to provide passive protection against the toxin (Li et al (2005) JVirol 79:10003-10012).

Other examples of antigens that can be displayed on the pili tip of aGram-positive bacterium according to the presently disclosed methodsinclude a mutant nontoxic form of the heat labile toxin LT A or LT Bproteins, and CooD, an ETEC adhesin, which are useful in providingprotection against enterotoxigenic Escherichia coli (ETEC). In someembodiments, the mutant LT A protein comprises a triple LT mutant (R7K,S63K, V53E) in which three residues required for toxin activity havebeen changed in ways that don't alter the protein structure (Pizza et al(1994) J Exp Med 180:2147-2153; the nucleotide and amino acid sequencesof the LT A protein are set forth in SEQ ID NO: 95 and 96, respectivelyand the nucleotide and amino acid sequences of the LT B protein are setare set forth in SEQ ID NO: 93 and 94, respectively). In thoseembodiments, wherein the antigen comprises CooD, the cooD gene from aCS1 ETEC strain (the nucleotide and amino acid sequence of which is setforth in SEQ ID NO: 89 and 90, respectively) can be used and itschaperone gene cooB will also be introduced into the Gram-positivebacteria displaying the antigen (Voegele, Sakellaris & Scott (1997) ProcNatl Acad Sci USA 94:13257-13261).

Vaccine delivery or immunization via attenuated bacterial vector strainsexpressing distinct antigenic determinants against a wide variety ofdiseases is now commonly being developed. Recently, mucosal (for examplenasal or oral) vaccination using such vectors has received a great dealof attention. For example, both systemic and mucosal antibody responsesagainst an antigenic determinant of the hornet venom were detected inmice orally colonized with a genetically engineered human oral commensalStreptococcus gordonii expressing the antigenic determinant on itssurface (Medaglini et al. (1995) Proc Natl Acad Sci USA 2:6868-6872).Also, a protective immune response was elicited by oral delivery of arecombinant bacterial vaccine wherein tetanus toxin fragment C wasexpressed constitutively in Lactococcus lactis (Robinson et al. (1997)Nature Biotechnology 15:653-657). Mucosal immunization as a means ofinducing IgG and secretory IgA antibodies directed against specificpathogens of mucosal surfaces is considered an especially effectiveroute of vaccination. In addition, the existence of a common mucosalimmune system permits immunization on one specific mucosal surface toinduce secretion of antigen-specific IgA and other specific immuneresponses at distant mucosal sites. Thus, in some of these embodiments,the Gram-positive bacteria that display an antigen comprise attenuatedpathogenic bacteria.

An alternative approach avoids the use of attenuated bacterial strainsthat may become pathogenic themselves by using recombinant commensalbacteria as vaccine carriers, such as Streptococcus spp. and Lactococcusspp (see, for example, Buccato et al. (2006) Journal of InfectiousDiseases 194:331-340). In some embodiments, the Gram-positive bacteriathat display an antigen comprise live, non-pathogenic bacteria.Non-limiting examples of non-pathogenic Gram-positive bacteria usefulfor the development of vaccines include Streptococcus gordinii,Staphylococcus xylosus, and Staphylococcus carnosus. Non-pathogenicbacteria can include, but are not limited to food-grade bacteria. Anon-limiting example of a food-grade bacterium is Lactococcus lactis.Lactococcus lactis is currently used as a probiotic and has beenreported to have adjuvant properties. Although it can colonize theintestines temporarily, it is not normally found in the humanmicroflora. Further, it is likely that a continuous cold chain would notbe required for delivery of an L. lactis vaccine and it would beinexpensive to produce. See Raha et al. (2005) Appl Microbiol Biotechnol68:75-81, which is herein incorporated in its entirety, for a review onthe use of L. lactis as a vaccine vector)

The presently disclosed immunological compositions can be formulated fordelivery, i.e., administering to the subject, by any available routeincluding, but not limited, to parenteral (e.g., intravenous),intradermal, subcutaneous, oral, nasal, bronchial, opthalmic,transdermal (topical), transmucosal, rectal, and vaginal routes. In someembodiments, the route of delivery is intravenous, parenteral,transmucosal, nasal, bronchial, vaginal, or oral.

The presently disclosed compositions also can include a Gram-positivebacterium with a pharmaceutically acceptable carrier. As used herein theterm “pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds also can be incorporatedinto the compositions.

As one of ordinary skill in the art would appreciate, a presentlydisclosed pharmaceutical composition is formulated to be compatible withits intended route of administration. Solutions or suspensions used forparenteral (e.g., intravenous), intramuscular, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfate;chelating agents, such as ethylenediaminetetraacetic acid; buffers, suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity, such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). The composition should be sterile and should be fluid to theextent that easy syringability exists. In some embodiments, thepharmaceutical compositions are stable under the conditions ofmanufacture and storage and should be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Ingeneral, the relevant carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In someembodiments, isotonic agents, for example, sugars, polyalcohols, such asmanitol or sorbitol, or sodium chloride are included in the formulation.Prolonged absorption of the injectable formulation can be brought aboutby including in the formulation an agent that delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., polynucleotide) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. In certain embodiments,solutions for injection are free of endotoxin. Generally, dispersionsare prepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In those embodiments in whichsterile powders are used for the preparation of sterile injectablesolutions, the solutions can be prepared by vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionsalso can be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches, and the like can contain any of the followingingredients, or compounds of a similar nature: a binder, such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient,such as starch or lactose, a disintegrating agent, such as alginic acid,Primogel, or corn starch; a lubricant, such as magnesium stearate orSterotes; a glidant, such as colloidal silicon dioxide; a sweeteningagent, such as sucrose or saccharin; or a flavoring agent, such aspeppermint, methyl salicylate, or orange flavoring. Compositions fororal delivery can advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the presently disclosed compositionscan be delivered in the form of an aerosol spray from a pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. Liquid aerosols, dry powders,and the like, also can be used.

Systemic administration of the presently disclosed compositions also canbe by transmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration,detergents, bile salts, and fusidic acid derivatives. Transmucosaladministration can be accomplished through the use of nasal sprays orsuppositories. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art.

The compounds also can be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical or cosmetic carrier. The specification for the dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofindividuals. Guidance regarding dosing is provided elsewhere herein.

Depending on the route of administration, the agent may be coated in amaterial to protect it from the action of enzymes, acids and othernatural conditions which may inactivate the agent. For example,solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

To administer an agent by other than parenteral administration, it maybe necessary to coat the agent with, or co-administer the agent with, amaterial to prevent its inactivation. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) andtrasylol.

In some embodiments, the polypeptide of interest displayed on the tip ofGram positive bacterial pili comprises an enzyme. As used herein, anenzyme is any polypeptide that can catalyze a chemical reaction. Thus,the presently disclosed Gram-positive bacteria that display enzymes canbe used in methods requiring whole cell biocatalysts. Enzymes useful forthe presently disclosed methods and compositions include those enzymesthat are capable of degrading organic matter, those that are involved inthe production of biofuels, or those that find use in improving thenutritive quality of food products, such as probiotics.

For the production of ethanol, Gram positive bacterium expressing anenzyme at the pilus tip that catalyzes a step in the degradation ofplant materials such as starch, cellulosic, lignocellulosic materials orthe like can be added to the plant materials. Non-limiting examples ofenzymes useful for this purpose include starch degrading enzymes such asα-amylases (EC 3.2.1.1), glucuronidases (E.C. 3.2.1.131); exo-1,4-α-Dglucanases such as −amyloglucosidases and glucoamylase (EC 3.2.1.3),β-amylases (EC 3.2.1.2), α-glucosidases (EC 3.2.1.20), and otherexo-amylases; and starch debranching enzymes, such as a) isoamylase (EC3.2.1.68), pullulanase (EC 3.2.1.41), and the like; b) cellulases suchas exo-1,4-3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-β-D-glucanase (EC3.2.1.39), β-glucosidase (EC 3.2.1.21), endo-1,4-β-glucanase (EC3.2.1.4) and the like; c) endoglucanases such as endo-1,3-β-glucanase(EC 3.2.1.6); d) L-arabinases, such as endo-1,5-α-L-arabinase (EC3.2.1.99), α-arabinosidases (EC 3.2.1.55) and the like; e) galactanasessuch as endo-1,4-β-D-galactanase (EC 3.2.1.89), endo-1,3-β-D-galactanase(EC 3.2.1.90), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC3.2.1.23) and the like; f) mannanases, such as endo-1,4-β-D-mannanase(EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), α-mannosidase (EC 3.2.1.24)and the like; g) xylanases, such as endo-1,4-β-xylanase (EC 3.2.1.8),β-D-xylosidase (EC 3.2.1.37), 1,3-β-D-xylanase, and the like; h) otherenzymes such as α-L-fucosidase (EC 3.2.1.51), α-L-rhamnosidase (EC3.2.1.40), levanase (EC 3.2.1.65), inulanase (EC 3.2.1.7) and the like,enzymes capable of degrading maltose maltotriose and α-dextrins obtainedfrom the first degradation of starch, include maltases, α-dexitrinase,α-1,6-glucosidases, glucoamylases (α-1,4-glucan glucohydrolases), andthe like, and enzymes capable of modifying monosaccharides, such asglucose isomerase, invertase, and the like.

Methods for improving the nutritive quality of food products (e.g.,probiotics) include, but are not limited to, the addition of food-gradeGram-positive bacterium displaying an enzyme that assists in digestionof certain food products (e.g., carbohydrates) to food products foranimal consumption. Such supplemented food products are particularlyuseful for subjects that exhibit enzymatic deficiencies and are lessable to digest particular food products. In specific embodiments ofthese methods, the Gram-positive bacteria comprise lactic acid bacteria,which are bacteria that are capable of converting sugars, includinglactose and other carbohydrates, into lactic acid. Non-limiting examplesof lactic acid bacteria include bacteria from the genera Lactobacillusor Bifidobacterium. Non-limiting examples of Lactobacillus speciesuseful as probiotics include L. rhamnosus, L. reuteri, L. casei, L.acidophilus, L. bulgaricus, L. plantarum, L. salivarius, L. johnsonii,and L. helveticus. Non-limiting examples of Bifidobacterium include B.lactis, B. infantis, B. longum, B. animalis, and B. bifidum. Theaddition of lactic acid bacteria to food products is particularly usefulfor the administration of the food products to people with lactoseintolerance. Bacterial strains useful for probiotics are known in theart (see, for example, Sanders (2007) Functional foods & nutraceuticals;June 2007:pp. 36-41, which is herein incorporated by reference in itsentirety). Enzymes that are useful in improving the nutritive quality offood products (e.g., for human or other animals) are known in the artand can be expressed on the pili of Gram positive bacteria (e.g.,Lactobacillus, Bifidobacterium) using the methods described herein.Non-limiting examples of such enzymes include lactase, hemi-cellulase,and phytase.

In other embodiments, the polypeptide of interest that is displayed onthe Gram positive pili comprises a biosorbent. As used herein, abiosorbent comprises a polypeptide that specifically binds with asubstantially high affinity to a particular molecule. Non-limitingexamples of biosorbents include polypeptides with a cellulose-bindingdomain, or a metal-binding domain, such as a metallothionein or aphytochelatin.

Gram-positive bacteria displaying a biosorbent find use inbioremediation methods. Specifically, the presently disclosed subjectmatter provides for methods for removing a contaminant from acomposition (e.g., soil, water), wherein the method comprisesintroducing to the composition a Gram-positive bacterium with apolypeptide of interest displayed on the tip of the pilus, wherein thepolypeptide of interest comprises a biosorbent capable of specificallybinding to the contaminant or an enzyme capable of degrading thecontaminant.

As used herein, the term “contaminant” refers to any inorganic ororganic molecule that is not desirable in a particular composition(e.g., soil, water). Non-limiting examples of contaminants includeenvironmental chemicals, radioactive elements, bacteria or organisms,the byproduct of the growth of bacteria or organisms, decomposingmaterial, or waste. In some embodiments, the composition comprising thecontaminant is soil or water. In some of these embodiments, thecontaminant comprises a heavy metal. In these embodiments, thepolypeptide of interest comprises a biosorbent, wherein the biosorbentcomprises a metal binding polypeptide that specifically binds to heavymetals. In some of these embodiments, the metal binding polypeptidecomprises a metallothionein or a phytochelatin.

In other embodiments, the contaminant comprises an organic contaminant.In these embodiments, the heterologous polypeptide comprises an enzymecapable of degrading the organic contaminant. In some embodiments, theorganic contaminant comprises an organophosphate. In some of theseembodiments, the heterologous polypeptide comprises organophosphoroushydrolase (OPH).

As used herein, the term “removing” when referring to a contaminantmeans there is less than 99%, less than 98%, less than 97%, less than96%, less than 95%, less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, less than 1% or less of the contaminantremaining in the composition after the introduction of the bacteriumdisplaying the biosorbent or degrading enzyme relative to the samecomposition prior to its introduction.

The presently disclosed Gram-positive bacteria also find use indiagnostic methods, wherein the Gram-positive bacteria display adetection reagent, which is a peptide (including, but not limited to, anantibody or a fragment thereof) capable of specifically detecting adisease-associated protein or ligand. In some of these embodiments, thedisplayed peptide further comprises a detectable label (e.g., aradiolabel, a fluorescent label). The Gram-positive bacteria displayingthe detection reagent can be administered to a subject, followed bydetection of the bacteria through the detectable label attached thereto.In some of these embodiments, the Gram-positive bacteria that isdisplaying the detection reagent comprise attenuated pathogenic bacteriaor non-pathogenic commensal bacteria.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Cpa Pilins are Present on the Tip of Group AStreptococcus Pili

To study the covalent linkage of major and minor pilin subunitscatalyzed by the pilin polymerase SrtC2, an expression system inEscherichia coli has been established (Zähner, D. et al. (2008) J.Bacteriol. 190:527-35). In this system, the only GAS genes present arethose encoding SrtC2, the pilins T3 and Cpa, and the chaperone, SipA2.Because complete pili are not synthesized in E. coli, a large fractionof the total pilin protein consists of the low molecular weight pilinpolymers. This genetic approach has allowed the definition of the pilinresidues required for covalent linkage of these subunits.

In this work, the linkage of the minor pilin protein Cpa to the backboneprotein of T3 pili of GAS was investigated. The results indicate thatthe noncanonical CWSS motif in Cpa is required for its attachment to theT3 protein by SrtC2. Evidence is also provided that addition of Cpa toT3 requires the same lysine residue in T3 that is needed forpolymerization of T3 subunits. This implies that addition of Cpa to a T3subunit leaves only the C-terminus of this T3 subunit available foraddition of another subunit. Therefore, the results strongly suggestthat Cpa is located exclusively at the tip of the T3 pilus, and, basedon this, a model for biogenesis of these pili has been suggested.

Cpa is not Linked to the N-Terminus of T3

In previous analyses of T3 pilus polymerization in E. coli, apresumptive T3-Cpa heterodimer using an HA-tagged derivative of Cpa wasidentified (Zähner, D. et al. (2008) J. Bacteriol. 190:527-35). Thisprotein was encoded on pJRS1326, which also encodes SipA2, T3 and SrtC2,all derived from the M3 GAS strain AM3 (see FIG. 1). To identify theCpa(HA) monomer, pJRS1325, a plasmid derived from pJRS1326 by deletionof srtC2 was used. Monomeric Cpa(HA) appeared as a band of approximately75 kDa on western blots of whole cell lysates of TOP10/pJRS1325 boiledin SDS (Zähner, D. et al. (2008) J. Bacteriol. 190:527-35 and FIG. 2Alane 2). In the presence of SrtC2, a second strong band that reactedwith both anti-T3 and anti-HA antisera was visible (Zähner, D. et al.(2008) J. Bacteriol. 190:527-35 and FIGS. 2A and B lanes 3). Themolecular mass difference between this approximately 105 kDa band andthe monomeric Cpa(HA) band is approximately that of the mature T3protein (about 32 kDa). To confirm that the 105 kDa band is aheterodimer of Cpa(HA) covalently bound to T3, a whole cell lysate of E.coli TOP10/pJRS1326 was subjected to immunoprecipitation using an antiHA-antibody. The immunoprecipitate was boiled in SDS to dissociatenoncovalent protein interactions and separated by SDS-PAGE. The 105 kDaband (FIG. 2B, lane 3) was recovered from a SyproRuby-stained SDS gel,digested with trypsin and subjected to mass spectrometry (MS) forpeptide identification. Both T3 and Cpa(HA) peptides were present in theimmunoprecipitated sample (see Table 1).

TABLE 1 T3/Cpa peptides identified by mass-spectrometry^(a) CpaHA SEQ ID Start^(b) End^(b) Before^(c) Sequence NO: After^(c)  46 54 G^(d)AEEQSVPNK 47 Q 72 80 K GYPDYSPLK 48 T 87 93 K VNLDGSK 49 E 120 130 KKLEGTNENFIK 50 L 121 130 K LEGTNENFIK 51 L 137 148 R IEDGQLQQNILR 52 I149 158 R ILYNGYPNDR 53 N 164 176 K GIDPLNAILVTQN 54 A 193 202 KAFQQEETDLK 55 L 236 245 Y QLSIFQSSDK 56 T 271 282 K YPYDVPDYATEK^(e) 57T 289 297 R KYAEGDYSK 58 L 290 297 K YAEGDYSK 59 L 298 305 K LLEGATLK 60L 306 317 K LAQIEGSGFQEK 61 I 318 323 K IFDSNK 62 S 347 355 Y GVATPITFK63 V 365 376 K NKEGQFVENQNK 64 E 367 376 K EGQFVENQNK 65 E 450 460 KYTHVSGYDLYK 66 Y 467 475 R DKDADFFLK 67 H 469 475 K DADFFLK 68 H 494 501K TLTEAQFR 69 A 528 534 K GYHGFDK 70 L 594 603 K QAPIIPITHK 71 L 609 618K TVTGTIADKK 72 K T3 SEQ  ID Start End Before Sequence  NO:  After 29 38A^(d) ETAGVSENAK 73 L 76 86 K DGLEIKPGIVN 74 G 107 114 K STEFDFSK 75 V115 124 K VVFPGIGVYR 76 Y 125 130 R YTVSEK 77 Q 131 142 K QGDVEGITYDTK78 K 144 153 W TVDVYVGNK 79 E 154 161 K EGGGFEPK 80 F 191/296 202/308K/K KNVSGNTGELQK/ 81/82 E/R TDESADEIVVTNK^(f) 218 226 K KDQIVSLQK 83 G219 226 K DQIVSLQK 84 G 243 253 K LKNGESIQLDK 85 L 245 253 K NGESIQLDK86 L 254 261 K LPVGITYK 87 V 262 273 K VNEMEANKDGYK 88 T ^(a)Massspectrometry performed on trypsin-digested band isolated from SDS-PAGE(105 kDa) ^(b)numbering refers to the position in the sequence of thepreprotein ^(c)residue before/after the peptide cleavage site^(d)residue preceding the predicted signal peptide cleavage site in thepreprotein ^(e)HA-tag sequence indicated in bold letters ^(f)peptideslinked by intramolecular isopeptide bond; residues predicted to formbond are underlined.

Since the sample was boiled prior to separation by SDS-PAGE, it isconcluded that the 105 kDa band corresponds to a covalently linkedT3-Cpa heterodimer produced in E. coli in the presence of SrtC2.

Because a sortase forms an amide bond between the carboxyl group at theC terminus of one protein and an amino group of a second protein, itseemed possible that the Cpa protein was attached at the α-amino groupof the distal T3 subunit in the pilus. T3 is synthesized as a preproteinthat is predicted to be cleaved by the signal peptidase between alanine28 and glutamate 29 (Zähner, D. et al. (2008) J. Bacteriol. 190:527-35).Consistent with this, the peptide representing the N-terminus of themature T3 protein (E29-K38) was recovered by MS, while the first 28residues of the preprotein of T3 were not among the peptides seen (FIG.2C). Since trypsin is not expected to cleave between A28 and E29,recovery of peptide E29-K38 indicates that cleavage occurred in E. coli,and not during MS sample preparation. Therefore, recovery of theN-terminal peptide of T3 from the Cpa-T3 heterodimer demonstrates thatthe α-amino group at the T3 N-terminus is not bound to Cpa(HA).

The VPPTG Motif in the CWSS of Cpa is Required for Linkage of Cpa to T3

Because covalent linkage of Cpa to T3 requires the sortase family enzymeSrtC2, the motif at the start of the CWSS of Cpa was expected to berequired for this reaction. However, this motif, VPPTG, differs from thecanonical LPXTG motif found in substrates of the housekeeping sortaseSrtA, like the M protein. It also differs from the CWSS motif in T3(QVPTG), which is required for its polymerization by SrtC2 (Barnett, T.C. et al. (2004) J. Bacteriol. 186:5865-75). Therefore, studies wereinitiated to establish whether the VPPTG motif in the CWSS of Cpa isessential for linkage of Cpa to T3. LPSTG was substituted for the VPPTGmotif of Cpa to test the importance of this motif in formation of theCpa-T3 heterodimer.

The HA-tagged derivative of Cpa was used in the assay that had beenestablished in E. coli (Zähner, D. et al. (2008) J. Bacteriol.190:527-35) to investigate this reaction. The desired motif replacementwas constructed by site-directed mutagenesis of pEU7646 (FIG. 1). DNAsequencing was used to confirm the presence of the mutation in theresulting plasmid, pEU7904. The formation of the T3-Cpa(HA) heterodimerwas examined, as well as the T3-T3 homodimer, by analysis of extracts ofE. coli strains that had been boiled in SDS to disrupt non-covalentbonds. TOP10/pJRS1325, which lacks srtC2, was used to facilitateidentification of the monomeric form of Cpa (FIG. 3A lane 1) and T3(FIG. 3B lane 1). As expected, in extracts of strain TOP10/pEU7646,which encodes Cpa(HA) with the wild type CWSS, western blot analysisusing an anti-HA antibody showed bands corresponding to the sizes forboth the Cpa(HA) monomer (75 kDa) and the Cpa(HA)-T3 heterodimer (105kDa) (FIG. 3A lane 2). However, no Cpa(HA)-T3 heterodimer was detectedin extracts of the strain in which LPSTG replaced the VPPTG motif of theCpa CWSS (TOP10/pEU7904; FIG. 3A lane 3). Instead, only the Cpa(HA)monomer and its characteristic degradation product, migrating at about32 kDa, were present. To verify that the absence of the Cpa(HA)-T3 dimerwas due to the introduction of the mutation in the Cpa(HA) CWSS motif,and not to an undetected second mutation that might affect the functionof SipA2 or SrtC2, the same cell lysates were analyzed by western blotusing anti-T3 antiserum (FIG. 3B). In both TOP10/pEU7646 andTOP10/pEU7904 (FIG. 3B lanes 2 and 3), T3 dimers were present, whilethey were absent from the strain lacking SrtC2 (FIG. 3B lane 1). Thisindicates that in both plasmids, the genes required for pilus formationfunctioned normally. The presence of polymerized forms of T3, combinedwith the absence of the T3-Cpa(HA) dimer in strain TOP10/pEU7904,demonstrate that the VPPTG motif in the CWSS of Cpa is required forlinkage of Cpa to T3.

In addition to replacing the VPPTG motif of Cpa with the LPSTG sequence,the VPPTG sequence of the pJRS9550 plasmid was mutated to delete the“PTG” residues of the motif, leaving just “VP” (pJRS9597; FIG. 1). Thisplasmid was transformed into the heterologous serotype M6 GAS strainJRS4, which lacks the FCT-3 region containing the genes required for T3pilus production and thus does not express T3 pili (see FIG. 10). Avector control, pJRS9545 (derived from pJRS9508), which consists of thepReg696 backbone and the P23 promoter, was transformed into JRS4 as anegative control. Cell wall fractions of this strain and strainsJRS4/pJRS9550 (wt), JRS4/pJRS9554 (-SrtC2), and JRS4/pJRS9597 (VP) thathad been boiled in SDS to dissociate noncovalent bonds were examined forformation of the Cpa(HA)-T3 heterodimer and incorporation of Cpa(HA)into HMW T3 polymers using an anti-HA antibody (FIG. 4A, lanes 1-4).Although the high molecular weight (HMW) banding pattern characteristicof pili on Gram-positive bateria (Mora et al. (2005) Proc Natl Acad SciUS A 102:15641-15646; Zähner and Scott (2008) J Bacteriol 190:527-535)was detected in cell wall extracts of JRS4/pJRS9550, the positivecontrol, no pilus bands were visible in extracts of JRS4/pJRS9597 (theVP mutant) or of the SrtC2-control JRS4/pJRS9554. To be sure that SipA2and SrtC2 remained functional in the mutant, the production of pili incell wall fractions was also examined using anti-T3 (FIG. 4 B). Thepresence of high molecular weight (HMW) forms of T3 in the mutantextract indicated that lack of incorporation of Cpa into pili inJRS4/pJRS9597 is not due to a defect in polymerization of T3. Asexpected, HMW forms of T3 were present in cell wall extracts ofJRS4/pJRS9550 (positive control) but not in those of itsSrtC2-derivative.

The absence of HMW polymers containing Cpa(HA) in cell wall extracts ofthe mutant might result either from lack of polymerization or from lackof covalent attachment of the pili to the cell wall. If the latter werecorrect, pilus polymers should be present in the culture supernatant.Therefore, concentrated supernatants were analyzed for the presence ofHMW forms containing Cpa(HA) and polymerized T3 (FIG. 4 A, lanes 5-8 anddata not shown). As expected, pili containing T3 were present in theconcentrated supernatant and Cpa-containing pili were present in thesupernatant from the positive control strain. However, Cpa(HA) was notpresent in HMW pilus forms in the supernatant of the VP mutant. Theabsence of incorporation of Cpa into the pili when the VPPTG motif ispartially deleted indicated that the VPPTG motif at the start of theCWSS of Cpa is required for addition of Cpa to T3 pili.

The QVPTG Motif in the T3 CWSS is not Necessary for Formation of theT3-Cpa Heterodimer

Previously, it was shown that the QVPTG motif (SEQ ID NO: 9) in the CWSSof T3 is required for polymerization of T3 by SrtC2 (Barnett, T. C. etal. (2004) J. Bacteriol. 186:5865-75). To determine whether this motifis also needed for the formation of the Cpa(HA)-T3 heterodimer, it wasreplaced in pEU7646 with the canonical LPSTG motif (SEQ ID NO: 2; FIG.1: pEU7905). Whole cell lysates of E. coli TOP10/pEU7905 were examinedby western blots using anti-T3 antiserum and anti-HA antibody (FIGS. 3 Cand D). As described above for the Cpa CWSS motif replacementexperiments, TOP10/pJRS1325, which lacks srtC2, was used to identify themonomeric forms of T3 (FIG. 3C lane 1) and Cpa (FIG. 3D lane 1). Wholecell lysates of the control strain TOP10/pEU7646, which encodes T3 withthe wild type CWSS, showed bands corresponding to the T3 monomer, the T3dimer, and the T3-Cpa(HA) heterodimer (FIG. 3C lane 2) as well as theCpa(HA) monomer and the Cpa(HA)-T3 heterodimer (FIG. 3D, lane 2). Aspreviously found (Barnett, T. C. et al. (2004) J. Bacteriol.186:5865-75), replacement of the native CWSS motif in T3 by LPSTG (inTOP10/pEU7905) prevented formation of multimers of T3, although the T3monomer was still present (FIG. 3C lane 3). Most importantly, theT3-Cpa(HA) heterodimer was also visible in lysates of this strain (FIGS.3C and D lanes 3). Thus, although the T3 CWSS motif is necessary for thepolymerization of T3, it is not required for the formation of theT3-Cpa(HA) heterodimer.

Lysine Residues Forming Putative Intramolecular Bonds in T3 are notRequired for its Polymerization

To identify lysine residues in the T3 protein that might be involved inpilus formation, available sequences of the predicted pilus backboneproteins of the FCT-2, FCT-3 and FCT-4 regions of different GAS strainswere compared (FIG. 5). Sequence alignment revealed 6 fully conservedlysine residues that correspond to K43, K81, K100, K106, K173, and K191in the T3 protein. Using pEU7657 (FIG. 1), which expresses T3, SrtC2 andthe chaperone-like protein SipA2 (Zähner, D. et al. (2008) J. Bacteriol.190:527-35) as a template, each of these lysines were replaced withalanine and analyzed polymerization of T3 for each mutant in extracts ofE. coli.

T3 dimers, and usually trimers, were visible on western blots of thecell extracts of the mutants with K to A corresponding to K43, K81,K100, K106, and K191 developed with anti-T3 antiserum (FIG. 6A).Surprisingly, the monomeric and polymeric forms of T3 mutant proteinsK43A and K191A migrated with an increased apparent molecular massrelative to the wild type T3 protein (FIG. 6A, lanes 3 and 7). Thehomologs of K43 and K191 in T1, the homolog of T3 in the FCT-2 region,(FIG. 5), have been shown to be involved in intramolecular bonds (Kanget al. (2007) Science 318:1625-1628). Therefore, these two lysineresidues are predicted to participate in intramolecular isopeptide bondsin T3. In support of this, the trypsin fragments of the Cpa(HA)-T3heterodimer identified by MS analysis included one containing two T3peptides linked by an isopeptide bond between K191 and N307 (Table 1).

The formation of these intramolecular bonds would be prevented bysubstituting alanine for the glutamate residue catalyzing formation ofthis bond (Kang et al. (2007) Science 318:1625-1628) or the lysineresidue participating in the bond. Thus, it seems likely that thealtered running behavior of the mutant proteins is a result of lack offormation of the intramolecular bonds. In agreement with this, thedouble mutant K43A,K191A protein migrates even more slowly than eithersingle mutant protein (FIG. 6B). However, for both single mutants andfor the double mutant, dimers of T3 protein were present and trimerswere also visible on some gels (FIGS. 6A and 6B and data not shown).This indicates that if these lysine residues are essential forintramolecular bond formation, they are not a requirement forpolymerization of T3.

Lysine residue 173 of T3 is required for T3 polymerization

Multimeric forms of T3 were present for all mutants except one: K173A(FIG. 6C). In the K173A mutant (FIG. 6C, lane 3), the presence ofmonomeric T3, which has an apparent molecular mass of 32 kDa whenanalyzed by SDS PAGE, indicates that the mutant protein is expressed andis stable in this strain. Because it seemed possible that the charge onthe lysine was the reason it was required for T3 polymerization, asecond mutant in which K173 was replaced with arginine (R) wasconstructed (FIG. 1 : pEU7692). Extracts of the E. coli straincontaining this mutation also showed only T3 monomers (FIG. 6C, lane 4),indicating the importance of the lysine group for polymerization of theT3 pilin. As a control, the adjacent lysine, K174, was also replacedwith A (FIG. 1:pEU7908). As expected, this had no visible effect on T3polymerization (FIG. 6C, lane 5). This demonstration that K173 isessential for T3 polymerization is consistent with the recent X-raycrystalographic and MS analysis of the M1 major pilin (Kang et al.(2007) Science 318:1625-1628; see FIG. 5).

The role of K173 in T3 pilus formation was also examined in GAS.Previous studies (Zähner, D. et al. (2008) J. Bacteriol. 190:527-35) ofT3 polymerization in GAS utilized pJRS9536 (FIG. 1), which contains thesame DNA fragment from the GAS serotype M3 strain AM3 as used above inE. coli (plasmid pEU7657), except that an HA tag was added to the T3protein (Barnett, T. C. et al. (2004) J. Bacteriol. 186:5865-75). Thisplasmid and its derivatives were analyzed in the heterologous serotypeM6 GAS strain JRS4, which lacks the FCT-3 region containing the genesrequired for T3 pilus production. The high molecular weight (HMW)banding pattern, which is characteristic of pili on Gram-positivebacteria (Mora, M. et al. (2005) Proc. Natl. Acad. Sci. 102:15641-6;Zähner, D. et al. (2008) J. Bacteriol. 190:527-35) was detected withanti-HA antibody in western blots of cell wall extracts that had beenboiled in SDS to dissociate noncovalent bonds. Plasmid pJRS9536 was usedas a template for site-directed mutagenesis to introduce the mutationsK173A and K173R in T3(HA), resulting in plasmids pJRS9541 and pJRS9543,respectively (FIG. 1). As a control, a K81A mutation was introduced intoT3(HA) in plasmid pJRS9536 because this residue did not affect pilusformation in E. coli (FIG. 6A). This mutation had no effect on T3polymerization in GAS as expected, although the total amount of allforms of T3 in GAS was reduced in this mutant relative to its wild typeparent (FIG. 7, lane 4 vs 1). In GAS, replacing K173 with either A or Rresulted in a loss of HMW T3(HA) polymers, while the monomer remainedplentiful (FIG. 7, lanes 2 and 3 vs. lane 1), which is in agreement withthe results in E. coli. The weak protein band that migrated with anapparent mass of about 65 kDa, consistent with the expected molecularweight of a T3(HA) dimer, does not appear to be a precursor to pilusformation since no higher molecular weight bands were visible. All fourcell wall extracts also contained degradation products of approximately20 kDa (FIG. 7, lanes 1-4).

The absence of HMW T3 polymers in cell wall extracts of the K173A mutantmight result either from lack of polymerization or from lack of covalentattachment to the cell wall. If the latter were correct, pilus polymersshould be present in the culture supernatant. To determine whether theT3(HA) monomer and/or its polymers are released into the culture medium,concentrated supernatants were analyzed for the presence of T3(HA). Thesupernatant from GAS strain JRS4/pJRS9536, which expresses the HA-taggedT3 protein along with the rest of the genes needed for pilus synthesis,showed a HMW banding pattern similar to that seen with the cell wallextract from this strain (FIG. 7, lanes 1 and 5). The culturesupernatant of the K81A mutant (JRS4/pJRS9538) showed a weak, butdiscernable, HMW pattern, indicating that polymerization of the T3protein occurred (FIG. 7, lane 8), although there was less total T3(HA)relative to the amount released from the wild type parent (pJRS9536). Incontrast, no HMW bands were detected in culture supernatants of eitherthe K173A or K173R mutants of T3 (JRS4/pJRS9541 or JRS4/pJRS9543,respectively) (FIG. 7, lane 6 and 7). The culture supernatants of allfour strains contained a similar pattern of degradation products. Theadditional band of approximately 65 kDa seen in cell extracts (seeabove) was also present in supernatants from both the K173A and K173Rmutants. From the absence of HMW bands of T3 in both the cell wall andsupernatant it is concluded that K173 is essential for polymerization ofT3(HA).

Lysine Residue 173 of T3 is also Required for Attachment of Cpa(HA) toT3

To identify the lysine in T3 required for the formation of a covalentbond to Cpa(HA), the conserved lysines (FIG. 5) were replaced withalanine using plasmid pEU7646, which encodes Cpa(HA), SipA2, T3 andSrtC2 (FIG. 1). Following confirmation of the induced mutation bysequencing, plasmids were introduced into E. coliBL21-CodonPlus(DE3)-RIL, and the formation of T3-T3 homodimer and T3-Cpaheterodimer was analyzed by western blot of whole cell lysates. Themonomeric form of Cpa(HA) was identified in an extract from a strainlacking SrtC2 (FIG. 8A lane 1), as before. The Cpa(HA)-T3 heterodimerwas present in lysates from T3 mutants K43A, K81A, K100A, K106A, andK191A (FIG. 8A, lanes 3-7), although extracts from mutants K81A andK106A appeared to contain less heterodimer. However, the lysate frommutant K173A showed no heterodimer, while K174A, used as a furthercontrol, produced the Cpa(HA)-T3 complex (FIG. 8B). This demonstratesthat lysine 173 of T3 is not only required for polymerization of T3, butis also needed for attachment of Cpa(HA) to the T3 shaft protein in E.coli.

To demonstrate the role of K173 of T3 in attachment of Cpa to thegrowing pilus in GAS, the M3 pilus cluster regions from pJRS1325,pEU7646, pEU7687 and pEU7688 (FIG. 1) were cloned into thepReg696-derivative pJRS9508 (Barnett et al. (2007) J Bacteriol189:1866-73), resulting in plasmids pJRS9554, pJRS9550, pJRS9557 andpJRS9558 respectively. Production of T3 pili by JRS4/pJRS9550 wasconfirmed by electron microscopy (FIG. 10). A vector control derivedfrom pJRS9508, consisting of the pReg696 backbone and the P23 promoter,was also constructed (pJRS9545). To identify monomeric Cpa(HA) and showits incorporation into the high molecular mass pilus ladder, cell wallextracts of JRS4 with each of these plasmids were prepared, boiled inSDS, and analyzed by western blot using an anti-HA antibody. Since theseconstructs differ from those used above (FIG. 7) by encoding Cpa(HA) andan untagged version of T3, the same cell wall extracts were alsoanalyzed using an anti-T3 antibody to confirm that in this geneticcontext K173 is essential for polymerization of T3 and for formation ofthe T3-Cpa heterodimer in GAS (FIG. 9B). In addition, supernatants ofeach culture were concentrated 10-fold by TCA precipitation and analyzedby western blot as described above. Strain JRS4/pJRS9554, which lackssrtC2 was used as a control to identify the band corresponding tomonomeric T3 (FIG. 9B lanes 2 and 6) and monomeric Cpa(HA) (FIG. 9Alanes 2 and 6). Cell wall extracts and supernatants from strainJRS4/pJRS9550, encoding Cpa(HA), SipA2, T3 and SrtC2, show the HMW formsof the pilus ladder, indicating that Cpa is present in these species(FIG. 9A lanes 3 and 7). Bands corresponding to sizes expected formonomeric Cpa(HA), the Cpa(HA)-T3 heterodimer, the Cpa(HA)-(T3)²hetero-trimer and the Cpa(HA)-(T3)³ hetero-tetramer are also visible(FIG. 9A lanes 3 and 7). While the Cpa(HA) species mentioned above werealso visible in cell wall extracts and supernatants of JRS4/pJRS9558 (T3K174A) (FIG. 9A, lanes 5 and 9), the cell wall extract and supernatantfrom strain JRS4/pJRS9557 (T3 K173A) resembled that of the strainlacking SrtC2 (JRS4/pJRS9554): it lacked the prominent bands of theCpa-T3 heterodimer and higher order polymers observed in the wild typeand K174A mutant (compare FIG. 9A lanes 2 and 6 with lanes 4 and 8).Thus, K173 is required for attachment of Cpa to T3 in GAS as well as inE. coli.

Cpa(HA) is Located at the Pilus Tip

Next, the position of Cpa(HA) in the T3 pili expressed by GAS wasexamined using whole-bacteria, negative-stain transmission electronmicroscopy (EM) coupled with immunogold localization. As expected,strain JRS4 containing the vector-only control plasmid pJRS9545 lackedany detectable pilus fibers (FIG. 10, panel A and D). In contrast,strain JRS4/pJRS9550, encoding Cpa(HA), SipA2, T3, and SrtC2, producedabundant surface fibers (FIG. 10, panels B, C, E, and F). The identityof the surface fibers were identified by incubating the bacteria withanti-T3 rabbit polyclonal antiserum, followed by detection with asecondary anti-rabbit antibody conjugated to 12-nm diameter goldparticles. The fibers produced by JRS4/pJRS9550 were abundantly labeledby the gold particles (FIG. 10, panel B and C), whereas only a few straygold particles were visible on the EM grid with the vector controlstrain (FIG. 10, panel A). To localize Cpa(HA) in the T3 pili, a duallabeling analysis was performed. For these experiments, the 12-nm goldparticles were used to identify T3 pilins, and Cpa(HA) was detectedusing an anti-HA mouse monoclonal antibody followed by an anti-mousesecondary antibody conjugated to 18-nm diameter gold particles. Duallabeling of the vector control strain again resulted in the presence ofonly a few, non-specific gold particles (FIG. 10, panel D). However,with strain JRS4/pJRS9550, the larger, HA-specific gold particles couldclearly be seen at what appeared to be the tips of some of the pilusfibers labeled by the smaller, T3-specific gold particles (FIG. 10,panels E and F). The EM data suggests that Cpa(HA) can be located at thepilus tips.

Discussion Motif in the CWSS

Like other proteins covalently linked to the cell wall of Gram-positivebacteria, pilins contain a CWSS at their C termini. Because the enzymerequired for pilin polymerization is a member of the sortase family oftranspeptidases, it is expected to behave like sortases, which areresponsible for covalent attachment of surface proteins to the cellwall. These enzymes recognize the motif at the beginning of the CWSS,usually LPXTG, cleave the substrate protein between the T and G andattach the T residue to an amino group of a second substrate. Unlike allthe pilins of the three serologically different Corynebacteriumdiphtheriae pili, as well as pili of S. pneumoniae, S. agalactiae, andBacillus cereus,which contain the canonical LPXTG motif, some pilins ofStreptococci contain non-canonical motifs in their CWSSs (Scott, J. R.et al. (2006) Mol. Microbiol. 62:320-30). For the T3 pilus of GAS, notonly is the motif of each of the three pilins that form the pilusnon-canonical, but it differs for each of these proteins. Nevertheless,SrtC2 catalyzes both polymerization of T3 and association of the minorpilin, Cpa, with the T3 pilus shaft. It had previously been demonstratedthat substitution of the canonical LPSTG motif (SEQ ID NO: 2) for theQVPTG motif (SEQ ID NO: 9) found in the shaft protein, T3, prevents itspolymerization (Barnett, T. C. et al. (2004) J. Bacteriol. 186:5865-75).The minor pilin, Cpa, has now been examined and it was found thatsubstituting LPSTG (SEQ ID NO: 2) for VPPTG (SEQ ID NO: 10) in thisprotein prevents its attachment to the shaft protein. In addition, adeletion within the VPPTG motif (SEQ ID NO: 10) in this protein preventsits attachment to the shaft protein, highlighting the necessity of aspecific motif at the start of the CWSS for this minor pilin. Thus, itappears that the two different motifs in T3 pilins (XXPTG; SEQ ID NO:11) are both recognized by SrtC2.

The Second Partner in the Intermolecular Bond Between T3 subunits

For formation of the peptide bond, the pilin polymerase must recognize aspecific motif N-terminal to the CWSS in the second pilin substrate,since the CWSS is cleaved and removed from the pilin. In C. diphtheriaeSpaA, SpaD and SpaH pilins, Ton-That and Schneewind identified aconserved “pilin motif” WxxxVxVYPK (SEQ ID NO: 97; Ton-That, H. et al.(2003) Mol. Microbiol. 50:1429-38) that plays this role. Bysite-specific mutagenesis, they showed that the K at the end of thismotif is required for pilin polymerization, and later demonstrated thatthis motif, together with the CWSS, is sufficient to cause an unrelatedS. aureus surface protein to be incorporated into SpaA pili (Ton-That,H. et al. (2004) Mol. Microbiol. 53:251-61.). They suggested, therefore,that the ε-amino group of this K participates in the peptide bond. Asimilar pilin motif is recognizable in pilins of some otherGram-positive bacteria, but not in all known or putative pilins. It isnot present in any of the proteins that constitute GAS pili.

For the GAS T1 major pilin protein, the K that is linked to the T of theCWSS motif of the next T1 subunit was recently identified by structuralanalysis (Kang, H. J. et al. (2007) Science 318:1625-8). Thecorresponding residue in the homologous T3 pilin is K173, as shown bysequence alignment (FIG. 4). In this work, it was demonstrated thatsubstitution of an A or an R for this residue abrogates T3polymerization, thus providing biological confirmation of theconclusions of Kang et al. that this bond is required for piluspolymerization.

The presence of two intra-molecular isopeptide bonds within the Ti shaftprotein was discovered recently by Kang et al. (Kang, H. J. et al.(2007) Science 318:1625-8). Each of these bonds is formed between theε-amino group of a lysine residue and the carboxyl group of anasparagine (N) residue within the same protein. By site-specificmutagenesis, they demonstrated that a glutamate (E) residue located neareach of the two intramolecular bonds is required for formation of thislink in a reaction that appears to be spontaneous. Since the wild typeT1 protein is more resistant to trypsin digestion than is the mutantprotein lacking intramolecular bonds, Kang et al. suggested that therole of the intramolecular bonds might be similar to that of disulfidebonds commonly found in pilins of Gram-negative bacteria i.e. they mightstabilize the folded protein and make it more resistant to forces itmight encounter in nature. The importance of these intramolecularpeptide bonds is suggested by conservation of the residues (KEN)required for their formation in the T1 protein in other pilus backboneproteins (FIG. 5). The MS analysis of the Cpa(HA)-T3 heterodimer(Table 1) identified one of the peptide fragments of T3 formed by anintramolecular isopeptide bond between K191 and N307, as predicted byhomology with the T1 protein (Kang et al. (2007) Science 318:1625-1628).The inability to identify a peptide containing the second predictedintramolecular bond should not be regarded as significant because the MScoverage of the T3 protein was limited. It was found, however, thatsubstitution of an A for either or both of the K residues predicted toparticipate in intramolecular bonds does not prevent T3-T3polymerization or addition of Cpa to T3. Thus, if these K residues areessential for formation of these bonds, then the intramolecular peptidebonds are not required for pilus biogenesis, although they may stillplay a role in the biological function of the pili.

Linkage of Cpa to T3

Attachment of Cpa to T3 is catalyzed by the same pilin polymerase,SrtC2, as that required for linkage of T3 subunits to each other (3,43). This enzyme catalyzes formation of a bond between a T in the CWSSand the e-amino group of a lysine in the next pilus subunit. If linkageof Cpa to T3 proceeds by the same enzymatic mechanism, there are fouralternative models of integration of a minor pilin into the pilusstructure (FIG. 11), two of which have been proposed previously (Telfordet al. (2006) Nat Rev Microbiol 4:509-519). It is theoretically possiblefor Cpa to be located exclusively at the tip of the T3 polymer and forthe T in the CWSS motif of Cpa to be linked to the α-amino group of theN-terminal amino acid of T3 (FIG. 11, model A). Because the intactN-terminal peptide of the mature T3 protein in the Cpa-T3 heterodimerwas identified in our mass spectrometric analysis, this possibility canbe ruled out. It is also possible for the T of the CWSS of T3 to belinked to a K of Cpa. There are two variants of this model. In the oneshown in FIG. 11, Model B, Cpa is interspersed within the T3 polymer (amodel proposed by Telford et al (2006) Nat Rev Microbiol 4:509-519). Inthe other version of this model (not shown), a minor pilin is anchoreddirectly to the cell wall and forms the base of the pilus, as occurs inGBS and C. diphtheriae (Nobbs et al. (2008) Infect Immun 76:3550-3560;Mandlik et al. (2008) Trends Microbiol 16:33-40). If either variant ofModel B were correct for Cpa, the Cpa-T3 heterodimer should still beformed when: (i) the VPPTG (SEQ ID NO: 10) of Cpa is changed (top T3monomer linked to Cpa in Model B), or (ii) K173 of T3 is mutated toanother residue. However, the Cpa-T3 heterodimer for either of thesemutations was not seen. Therefore, Model B is unlikely to be correct forCpa. Model C assumes that Cpa is attached to the T3 polymer by a lysinein T3 different from K173, which links the T3 subunits to each other(FIG. 11, Model C; branched model proposed by Telford et al. (2006) NatRev Microbiol 4:509-519). In this case, as in Models A and B, K173 of T3would not be needed to form the Cpa-T3 heterodimer. Since it was foundthat a K173A mutation of T3 abolished formation of the Cpa-T3heterodimer, Model C can also be eliminated. The remaining possibilityis for the T of the CWSS of Cpa to be linked to T3 by K173 (FIG. 11,model D). This model predicts that mutation of K173 of T3 would preventformation of the Cpa-T3 heterodimer, which is what was found. Additionalsupport for this model is that replacement of the QVPTG motif (SEQ IDNO: 9) of T3 with LPSTG (SEQ ID NO: 2) did not prevent formation of thedimer with Cpa. In summary, it was found that the same K173 residue thatlinks T3 monomers to each other is required for attachment of Cpa to T3and the VPPTG motif of Cpa is needed for this attachment, while theQVPTG (SEQ ID NO: 9) of T3 is not required. Thus, model 8D appears to becorrect and it appears that Cpa can only be located on the T3 pilus tip.

Localization of Cpa in T3 Pili by Immunogold EM

Model D (FIG. 11) indicates that the minor pilin, Cpa, must be locatedat the pilus tip. This location was supported by our immunogold EManalysis of the T3 pili expressed by intact GAS bacteria. The electronmicrographs indicate that the pilus fibers protrude from the bacterialsurface and may well extend beyond the capsule surrounding the GASstrain. The T3 pili appeared as long, thin fibers that tended to twistand bundle together. Dual labeling of the bacteria to detect both themajor pilin T3 and minor pilin Cpa demonstrated the presence of Cpa atwhat appeared to be the pilus tips. This localization was consistentlyobserved, although quantitative analysis of Cpa localization was notpossible due to the flexible nature of the pili.

Location of Pilus Adhesin

The tip location of the Cpa minor pilin is similar to that found for theadhesin protein of pili on Gram-negative bacteria that are assembled bythe chaperone-usher or alternate chaperone-usher pathways (e.g. Pap andCS1 pili respectively). In these cases, distal location of the adhesinis generally considered to facilitate its interaction with the receptorto which the pilus attaches. However, for GAS, the role of Cpa inadherence of T3 pili or in adherence of the homologus Ti pili is notclear. Abbot et al. (Abbot, E. L. et al. (2007) Cell Microbiol.9:1822-1833) have shown that in a strain producing T1 pili, these piliare required for adherence to primary human keratinocytes or humantonsilar epithelial cells, which are likely to represent the cells towhich GAS must attach for initiation of infection. However, in thisstrain, pili are not needed for attachment to A549 or HEp-2 cells. TheCpa protein of a serotype M49 GAS strain, which has an FCT-3 pilus locussimilar to that of the T3 pilus, has been found to bind to type 1collagen, an important extracellular matrix protein in the human hostand to mediate adherence to HEp-2 cells (Kreikemeyer, B. et al. (2005)J. Biol. Chem. 280:33228-39). However, for the M1 GAS strain studied byKehoe's group, collagen binding does not appear to be important foradherence to primary human keratinocytes or human tonsillar epithelialcells, since preincubation of either type of human cell with collagendid not affect GAS adherence (Abbot, E. L. et al. (2007) Cell Microbiol.9:1822-1833).

The adhesin for Streptococcus pneumoniae (Nelson, A. L. et al. (2007)Mol. Microbiol. 66:329-40), S. agalactiae (Dramsi, S. et al. (2006).Mol. Microbiol. 60:1401-13; Krishnan, V. et al. (2007) Structure15:893-903.) and C. diphtheriae (Mandlik, A. et al. (2007) Mol.Microbiol. 64:111-24) is a minor pilin protein, encoded by the firstgene in the pilus locus, and the pilus shaft protein is dispensable foradherence to the cells studied. In contrast, for the T1 pili of the M1GAS strain, all three pilin proteins are required for adherence:deletion of the genes for any of the three pilin proteins preventedadherence (Abbot, E. L. et al. (2007) Cell Microbiol. 9:1822-1833).However, it is still possible that Cpa is a specific adhesin of T1 andT3 pili, since the shaft protein may be required only to present theadhesin so that it is external to the cell capsule. The role andlocation of the other minor pilin for T1 or T3 pili has not beeninvestigated yet. It may be interspersed along the shaft of the pilus(Model 8C) as occurs for pilins in S. agalactiae (Rosini, R. et al.(2006) Mol. Microbiol. 61:126-41) and S. pneumoniae (Barocchi, M. A. etal. (2006) Proc. Natl. Acad. Sci. 103:2857-62; Hilleringmann, M., et al.(2008) PLoS Pathog. 4:e1000026), or it may be located exclusively at thetip in place of Cpa on some T3 pili. The latter location would producepili with different specificities on the same bacterial cell.

Comparison with Pili of Gram-Negative Bacteria: Model for Pilus Assembly

Unlike the much larger flagellae in which new subunits are transportedthrough the structure and added at the tip (Macnab, R. M. et al. (2003)Annu. Rev. Microbiol. 57:77-100), pili on Gram-negative bacteria growfrom the base out. In the well-studied Pap pili, the tip protein isadded first and serves to nucleate formation of the pilus structure(reviewed by Sauer, F. G. et al. (2004) Biochim. Biophys. Acta1694:259-67; Thanassi, D. G. et al. (2005) Mol. Membr. Biol. 22:63-72).This is accomplished by the strong affinity of a tip-chaperone complexfor the usher protein, which forms a pore in the outer membrane of theGram-negative cell. Interaction with the usher is proposed to alter theconfiguration of the tip-chaperone complex so that a shaft subunit cannow displace the tip protein from the usher to allow addition of furthersubunits, leading to continued pilus growth.

Although assembly of pili on Gram-positive bacteria requires a specificpilin polymerase, in both Gram-positive and Gram-negative bacteria, theSec system is used to transport pilins across the membrane of the cell.It is likely that pili on Gram-positive bacteria also grow by adding newsubunits from the bottom because, based on the presence of a predictedmembrane-spanning domain, the pilin polymerase is expected to bemembrane located. However, the minor pilins are not required to nucleateformation of the pilus structure since they are dispensable forformation of pili in C. diphtheriae, S. agalactiae, S. pneumoniae andGAS. It was found that the minor pilin, Cpa, is likely to be locatedexclusively at the T3 pilus tip, therefore it must be added first as thepilus grows. In agreement with this idea, Cpa is found in all the HMWbands of growing pili in GAS. Ordered subunit incorporation might beaccomplished by a mechanism involving differential affinity, similar tothat used for Pap pilus assembly. The membrane-located “gating” proteinin Pap pili is the usher, while in Gram-positive bacteria it would bethe pilin polymerase. This assembly model predicts that for GAS T3 pili,the polymerase SrtC2 will be found to have a greater affinity for Cpathan for T3. The relative abundance of the Cpa-T3 heterodimer vs. the T3homodimer in FIG. 3C lane 2 is in agreement with this prediction. Whenmore Cpa becomes available in the cell, growth of a new pilus should beinitiated as long as there is an excess of SrtC2 and all other sites andproteins (e.g. SipA2) required. This would lead to limitation of growthof old pili. Thus, regulation of synthesis of the pilin proteins shouldbe important in determining the length and number of pili on the GASsurface.

In summary, the residues required in Cpa and T3 for SrtC2-catalyzedpeptide bond formation have been identified. It was also learned thatthe K residues that appear to be involved in formation of the recentlydescribed intramolecular peptide bonds in the shaft protein of the T3pilus are not required for pilus polymerization, suggesting that theintramolecular peptide bonds are not needed for this process. Finally,because it was found that K173 of T3 is required for addition of Cpa aswell as for T3-T3 polymerization, it is likely that Cpa is locatedexclusively at the tip of the T3 pilus structure.

Materials and Methods for Experimental Example 1 Bacterial Strains andGrowth Conditions

GAS strain JRS4 is a spontaneous streptomycin-resistant derivative ofthe serotype M6 strain D471 (32). GAS strains were grown in Todd-Hewittmedium supplemented with 0.2% yeast extract (Difco). E. coli strainsTOP10 (Invitrogen) and BL21-CodonPlus(DE3)-RIL (Stratagene) were grownin Luria broth (LB) (30). Antibiotics were used in the followingconcentrations: kanamycin 50 μg/ml and spectinomycin 100 μg/ml. IPTG ata final concentration of 1 mM was used for induction.

Site Specific Mutagenesis

Mutagenesis was performed using the QuikChange II XL mutagenesis Kit(Stratagene) according to the manufacturer's protocol using the primersshown in Table 51. Mutagenized plasmids were transferred into E. coliBL21-CodonPlus(DE3)-RIL (Stratagene) or TOP10 (Invitrogen). Correctnucleotide replacement was confirmed by DNA sequencing of themutagenized gene.

Preparation of Cell Lysates and Cell Wall Extracts

Cell lysates of E. coli were obtained from overnight cultures grown withantibiotics and IPTG if appropriate. Samples were prepared from E. coliand GAS as described previously (Zähner, D. et al. (2008) J. Bacteriol.190:527-35).

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andImmunoblot analysis: Proteins were separated by SDS-PAGE on 4 to 12%gradient gels (NuPAGE, Invitrogen) and transferred to nitrocellulosemembrane (BioRad) for immunoblot analysis. The monoclonal anti-HAantibody (clone HA-7, Sigma) was used at a 1:2,000 dilution. Thepolyclonal anti-T3 antiserum, used at a 1:250 dilution is a T3 typingserum provided by Dr. B. Beall (CDC, Atlanta). T3 typing sera have beendemonstrated to cross-react with Cpa and other proteins encoded in theFCT region (15).Immunoprecipitation of Cpa(HA)-T3. E. coli

Top10/pJRS1326 cells were grown to OD_(600nm) of 1.2, and the cellpellet resuspended in 1/50 volume of RIPA buffer (150 mM NaCl, 1.0%Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate[SDS], 50 mM Tris; pH 8.0). Cells were disrupted by sonication, andinsoluble cell debris was removed by centrifugation at 12,000×g for 15min at 4° C. Immunoprecipitation of crude cell extracts was performedusing “EZview Red Anti-HA Affinity Gel” (Sigma) according to themanufacturer's protocols. The immunoprecipitated protein was subjectedto SDS-PAGE on a 4 to 12% gradient gel (NuPAGE, Invitrogen) followed byvisualization with SYPRORuby (Invitrogen) according to themanufacturer's instructions. The band migrating with an apparentmolecular weight of 105 kDa, corresponding to the Cpa(HA)-T3 heterodimerwas excised, and stored at 4° C. until analysis by mass spectrometry.

Peptide Preparation and Mass Spectrometry

The excised protein band was subjected to trypsin digestion and massspectrometric analysis (MALDI-TOF-MS/MS analysis) (Medzihradszky, K. F.et al. (2000) Anal. Chem. 72:552-8; Venkataraman, N. et al. (2005) J.Immunol. 175:7560-7) at the microchemical and proteomics facility atEmory University as described previously (Freeman, W. M. et al. (2005)Pharmacogenomics J. 5:203-14; Tseung, C. W. et al. Biochem J.380:211-8). GPS Explorer 2.0 software (Applied Biosystems) and a MASCOT(www.matrixscience.com/) search engine were used for identification ofpeptide fragments. The National Center for Biotechnology Informationnonredundant database was used for the searches.

Electron Microscopy

For immunogold-EM, S. pyogenes strains JRS4/pJRS9545 (vector control) orJRS4/pJRS9550 (expressing T3 pili with Cpa(HA)) were grown as describedabove, harvested, washed with PBS, and then adsorbed to polyvinylformal-carbon-coated grids (E.F. Fullam, Inc.) for 2 minutes and fixedwith 1% glutaraldehyde for 1 minute. For single labeling experiments,the grids were washed twice with PBS, blocked with PBS+1% BSA, and thenincubated for 1 hour with a 1:200 dilution (in PBS+1% BSA) of the rabbitpolyclonal anti-T3 antiserum described above. The grids were washedthree times with PBS and then incubated for 1 hour with a 1:50 dilution(in PBS+1% BSA) of anti-rabbit IgG antibody conjugated to 12-nm diametercolloidal gold particles (Sigma-Aldrich). The grids were washed threetimes with PBS and twice with water, and then negatively stained with0.5% phosphotungstic acid (Ted Pella, Inc.) for 35 seconds. For duallabeling experiments, grids prepared as described above were thenincubated for 1 hour with 1:50 dilutions of both the anti-T3 antiserumand the anti-HA mouse monoclonal antibody described above. The gridswere washed three times with PBS and then incubated for 1 hour with 1:50dilutions of both the 12-nm gold anti-rabbit IgG antiserum and ananti-mouse IgG antibody conjugated to 18 nm diameter colloidal goldparticles (Sigma-Aldrich). The grids were then washed and stained asdescribed above. The grids containing the negatively stained bacteriawere examined on an FEI TECNAI 12 BioTwin G02 microscope (FEI) at 80 kVaccelerating voltage. Digital images were acquired with an AMT XR-60 CCDdigital camera system (Advanced Microscopy Techniques).

Table 2 presents information on the primers that were used in thesestudies.

TABLE 2 Primers used in these experiments^(A). SEQ  ID Sequence PrimerNO: (5′-3′) Plasmids^(B) Orf100K43A_F 16 CCGAAAATGCAAAATpEU7646→pEU7652, TAATAGTAAAAgctA  pEU7657→pEU7678 CATTTGACTCTTAT ACAGACOrf100K43A_R 17 GTCTGTATAAGAGTC pEU7646→pEU7652, AAATGTagcTTTTACpEU7657→pEU7678 TATTAATTTTGCATT TTCGG Orf100K81A-F 18 CGAAAGACGGTTTAGpEU7646→pEU7651, AGATTGCTCCAGGTA pEU7657→pEU7679 TTGTTAATGGTTTAA CAGOrf100K81A-R 19 CTGTTAAACCATTAA pEU7646→pEU7651, CAATACCTGGAGCAApEU7657→pEU7679 TCTCTAAACCGTCTT TCG Orf100K100A_F 20 CAGCTATACTAATACpEU7646→pEU7653, TGATgcaCAGATAGT pEU7657→pEU7679 AAAGTTAAAAGTACA GAGOrf100K100A_R 21 CTCTGTACTTTTAAC pEU7646→pEU7653, TTTACTATCTGGtgcpEU7657→pEU7679 ATCAGTATTAGTATA GCTG Orf100K106A_F 22 CTGATAAACCAGATApEU7646→pEU7654, GTAAAGTTgcaAGTA pEU7657→pEU7681 CAGAGTTTGATTTTT CAAAAGOrf100K106A_R 23 CTTTTGAAAAATCAA pEU7646→pEU7654, ACTCTGTACTtgcAApEU7657→pEU7681 CTTTACTATCTGGTT TATCAG Orf100K173A_F 24 CTAAGGAACAAGGApEU7646→pEU7687, ACAGACGTCgcAAAA pEU7657→pEU7907 CCAGTTAATTTTAAC AACOrf100K173A_R 25 GTTGTTAAAATTAAC pEU7646→pEU7687, TGGTTTTcgGACGTCpEU7657→pEU7907 TGTTCCTTGTTCCTT AG Orf100K173R_F 26 CTAAGGAACAAGGApEU7657→pEU7692 ACAGACGTCcgAACC AGTTAATTTTAACAA C Orf100K173R_R 27GTTGTTAAAATTAAC pEU7657→pEU7692 TGGTTTTcgGACGTC TGTTCCTTGTTCCTT AGOrf100K174A_F 28 CTAAGGAACAAGGA pEU7646→pEU7688, ACAGACGTCAAAgcApEU7657→pEU7908 CCAGTTAATTTTAAC AAC Orf100K174A_R 29 GTTGTTAAAATTAACpEU7646→pEU7688, TGGTgcTTTGACGTC pEU7657→pEU7908 TGTTCCTTGTTCCTT AGOrf100K191A_F 30 GCAACTACTTCGTTA pEU7646→pEU7661, AAAGTTAAGgcaAAT pEU7657→pEU7682, GTATCGGGGAATACT  pEU7678→pEU7909 GG Orf100K191A_R 31CCAGTATTCCCCGAT pEU7646→pEU7661, ACATTtgcCTTAACT pEU7657→pEU7682,TTTAACGAAGTAGTT pEU7678→pEU7909 GC Cpa_LPSTG_Sense 32 GAAAACCGAAAAGApEU7646→pEU7904 TCTTcTCCCAtCAA CTGGTTTGACAACA GATGG Cpa_LPSTG_Anti 33CCATCTGTTGTCAAA pEU7646→pEU7904 CCAGTTGaTGGGAgA AGATCTTTTCGGTTT TCT3_LPSTG_Sense 34 GTCACAAATAAGCGT pEU7646→pEU7905 GACACTCtACCttCAACTGGTGTTGTAGGC ACCCTTGCTCC T3_LPSTG_Anti 35 GGAGCAAGGGTGCCpEU7646→pEU7905 TACAACACCAGTTGa AggTaGAGTGTCACG CTTATTTGTGACCpa_VP1_Sense 45 GAAAACCGAAAAGA pJRS9550→pJRS9597 TCTTGTCCCATTGACAACAGATGG Cpa_VP1_Anti 46 CCATCTGTTGTCAAT pJRS9550→pJRS9597GGGACAAGATCTTTT CGGTTTTC ^(A)Uppercase letters represent basescomplementary to GAS sequence. Lowercase letters represent bases addedor changed to facilitate cloning or mutagenesis. ^(B)Templates used inPCR with according primer pairs, and the resulting plasmids.

Example 2 Expression of a Polypeptide Fused to Cpa in E. coli

Constructs containing a polynucleotide encoding a fusion proteincomprising the maltose binding protein (encoded by the malE gene) andamino acid residues 594-744 of Cpa (SEQ ID NO: 6) were transformed intoE. coli strain XL10. The fusion protein further comprised anamino-terminal Sec-dependent signal sequence. Constructs used in thisstudy were pJRS9555 (comprises the FCT-3 region from M3 GAS strain AM3from the MBP/Cpa gene through SrtC2) or pJRS9556 (comprises the sameFCT-3 region from M3 GAS strain AM3 from the MBP/Cpa through T3), whichare derivatives of the pJRS1326 construct (see FIG. 1). Cell lysates andcell wall extracts were prepared as described in Zähner and Scott (2008)J Bacteriol 190:527-535. The extracts were treated with hot SDS todissociate molecules that are not covalently linked. Immunoblot analysiswith an anti-MBP and anti-T3 antibody was performed as described inExperimental Example 1 and results are shown in FIG. 12. Lanes 3, 6, and7 have the genes needed to link the MBP/Cpa fusion protein to thegrowing T3 polymer (MBP/Cpa, T3, SipA, and SrtC2). In lanes 3, 6, and 7MBP/Cpa is covalently linked to T3. Lanes 8, 9, and 10 are negativecontrols lacking the gene encoding the enzyme that catalyzes thisprocess (SrtC2). Lanes 3, 6, and 7 were confirmed by PCR analysis tohave the desired insert; lanes 2, 4, and 5 lack the insert and lanes8-10 lack srtC2. Therefore, the MBP/Cpa fusion protein can be added tothe T3 polymer in E. coli.

Example 3 Expression of a Polypeptide Fused to Cpa in Lactococcus lactis

A plasmid, referred to herein as pJRS9565, was constructed whichcomprises the FCT-3 region from the M3 GAS strain AM3 including the Cpagene, SipA2, T3, and SrtC2 (see FIG. 13), wherein the gene encodingmaltose binding protein was inserted within the Cpa gene. The plasmidthus encodes a MBP/Cpa fusion protein which is referred to herein asMBP*. The nucleotide and amino acid sequences of the MBP/Cpa fusion areset forth in SEQ ID NOs: 98 and 99, respectively. The nucleotidesequence comprises 30 nucleotides in the 5′ untranslated region of Cpa,followed by the first 56 codons of the coding sequence, the MBP codingsequence, and then the nucleotide sequence encoding amino acid residues594-744 of Cpa. This construct comprises the coding sequence for theSec-dependent signal peptide sequence of Cpa (the amino acid sequence isset forth in SEQ ID NO: 100). Once the expressed protein istranslocated, the Sec-dependent signal peptide is cleaved, resulting ina MBP/Cpa fusion protein comprising the sequence set forth in aminoacids SEQ ID NO: 102. A control plasmid pJRS9566, which lacks SrtC2, wasalso constructed and used in the following studies.

Lactococcus lactis strain MG1363 was transformed with the pJRS9565 orthe control pJRS9566 plasmid and the exposure of the MBP* antigen and T3on the surface of intact MG1363/pJRS9565 was examined by whole cell dotblot with a monoclonal anti-MBP antibody and polyclonal anti-T3antiserum. The MBP* antigen and T3 are both surface exposed inMG1363/pJRS9565 as demonstrated by reaction with the anti-MBP antibodyand anti-T3 antiserum (FIGS. 14A and 14B, lanes 1-4, rows E, F (+)). Asexpected, MG1363/pJRS9566, which lacks SrtC2, does not react with eitherof these antibodies (FIGS. 14A and 14B lanes 1-4, row G (-SrtC)).

To examine whether the MBP* antigen is incorporated into HMW polymerscharacteristic of pili in Gram-positive bacteria (Scott and Zähner(2006) Mol Microbiol 62:320-330; Telford et al. (2006) Nat Rev Microbiol4:509-519; Mandlik et al. (2008) Trends Microbiol 16:33-40), cell wallfractions of strains MG1363/pJRS9565 (MBP*), MG1363/pJRS9566 (-SrtC2),and MG1363/pJRS9545 (vector control) were prepared and analyzed bywestern blot with anti-MBP and anti-T3. The MBP*-T3 heterodimer (80 kDa)and the high molecular mass ladder characteristic of pili are seen incell wall extracts of MG1363/pJRS9565 analyzed with anti-MBP andanti-T3, indicating that MBP* is incorporated into the pilus structureand that T3 pilus polymerization occurs normally (FIGS. 15A and 15B,lanes 1 and 2). As expected, analysis of the cell wall fraction fromMG1363/pJRS9566 (SrtC2-) with anti-MBP and anti-T3 shows only themonomeric forms of MBP* and T3, with apparent molecular masses of 57 kDaand 32 kDa respectively (FIGS. 15A and 15B, lane3). No cross reactivitywith either the anti-MBP antibody or the anti-T3 antiserum is observedwith the cell wall fraction of MG1363/pJRS9545.

Negative-stain transmission electron microscopy of whole bacteriacoupled with immunogold localization (performed using similar methods asthose described in Experimental Example 1) reveals that abundant surfacefibers are expressed by MG1363/pJRS9565. Analysis with polyclonalanti-T3 antiserum followed by detection with a secondary anti-rabbitgold-conjugate antibody indicates that these fibers are composed of theT3 protein (FIGS. 16A and 16B). As expected, MG1363 containing thevector control does not express pili or react with the T3 antibody (FIG.16C).

To determine whether MBP* is synthesized in MG1363 in an active form,lysates of MG1363/pJRS9565 and MG1363/pJRS9566 were applied to amyloseresin, and the eluate, flow through and crude lysate fractions wereanalyzed by western blot with the anti-MBP antibody and the anti-T3antiserum for the presence of HMW pilus polymers. Lysates ofMG1363/pJRS9545 treated in the same fashion were used as a negativecontrol. HMW pilus forms are detected by both the anti-MBP antibody andthe anti-T3 antiserum in the eluate fraction of MG1363/pJRS9565,indicating that MBP remains active and confers the ability to bindamylose resin to hybrid pili (FIGS. 17A and 17B, lane 2). As expected,only the monomeric form of the MBP* is seen in the eluate fraction ofMG1363/pJRS9566 when analyzed in this fashion (FIGS. 17A and 17B, lane3). No cross reactivity with either the anti-MBP antibody or the anti-T3antiserum is observed with extracts of MG1363/pJRS9545.

To examine the possibility that the binding of MBP* pili to the amyloseresin is nonspecific in nature, lysates of MG1363/pJRS9550, whichproduces wild type (wt) T3 pili, were purified using the amylose resinand analyzed with anti-MPB and anti-T3 as described above. Duplicatesamples corresponding to the elution (E) fraction of MG1363/pJRS9565 andthe elution (E), flow through (F) and crude lysate fractions ofMG1363/pJRS9550 were transferred to nitrocellulose. The membrane was cutdown the middle (slide 10 lane 5) and half was analyzed with monoclonalanti-MBP antibody (FIG. 18, lanes 1-4) while the other half was analyzedwith monoclonal anti-HA antibody (FIG. 18, lanes 6-9). As expected, thepili produced by MG1363/pJRS9565 (MBP*) bind to the amylose resin andcan be eluted with maltose as HMW forms that react with anti-MBP (FIG.18, lane 1). In contrast, the pili of MG1363/pJRS9550 do not bind to theamylose beads, and are detected in the flow through and crude lysatefractions using the anti-HA antibody (FIG. 18, lanes 8, 9). These datademonstrate that the binding of hybrid pili to the amylose resin is nota result of interactions between the wild type pilus subunits and theamylose beads, but is rather due to the ability to bind amyloseconferred upon hybrid pili by MBP*.

Materials and Methods for Experimental Example 3

Strains, Plasmids and Growth Conditions Lactococcus lactis MG1363 wascultured without shaking at 30° C. in M17 media (OXOID) supplementedwith 0.5% glucose (GM17). MG1363 was made competent by the method ofHolo and Nes (Holo and Nes (1989) Appl Environ Microbiol 55:3119-3123).Spectinomycin was used at a concentration of 100 μg/mL.

Cell Wall Extraction

Cell wall fractions of MG1363 were obtained using a modification of theprocedure of Buccato et al. (2006) J Infect Dis 194:331-340, as follows.Overnight cultures of MG1363 were centrifuged at 4000 rpm for 10 minutesat 4° C. in an Eppendorf 5810R tabletop centrifuge with an A-4-62swinging bucket rotor. The pellet was resuspended in 1/10 volume ofsaline (0.9% m/v NaCl), transferred to a 1.5 mL Eppendorf tube, andcentrifuged at 13000 rpm for 1 minute at 4° C. in a Spectrafuge 16Mmicrocentrifuge. The pellet was resuspended in the same volume of salinesolution, and the optical density at 600 nm (OD₆₀₀) was determined at adilution of 1:100. The concentration of cells in cell units/mL [CU/mL]was calculated as previously described (Biswas et al. (2001) InfectImmun 69:7029-7038). Four CU was transferred to a new 1.5 mL tube, andcentrifuged as above. Cell wall extraction was performed in 160 μL oflysis buffer (50 mM Tris-HC16.8, 30% raffinose, Roche Complete proteaseinhibitors, 4 mg/mL lysozyme, 400 U/mL mutanolysin) at 37° C. for 3hours with gentle rotation. Samples were centrifuged at 13000 rpm for 1minute at room temperature, and the supernatant was transferred to a newtube, and recentrifuged at 13000 rpm for 4 minutes at room temperature.Then, 75 μL of the second supernatant was combined with 25 μL of 4×SDSsample buffer (Sambrook et al., 1989) in a new tube and samples wereheated to 100° C. for 10 min.

Dot Blot

Dot blot was performed by a slight modification of the procedure ofBiswas et al. (2001) Infect Immun 69:7029-7038. Briefly 5 μL of anovernight culture of MG1363 that had been washed in saline solution asdescribed above, was spotted onto a nitrocellulose membrane (Bio-Rad)and dried for 3 hours at room temperature. Membranes were blocked atroom temperature in blocking solution (3% BSA in TBS 7.6, 0.02% NaN₃)for 30 minutes with gentle orbital rotation, followed by analysis withthe appropriate antibody.

Amylose Purification of Hybrid Pili

Overnight cultures of MG1363 were centrifuged at 4000 rpm at 4° C. for10 minutes, and the pellets were resuspended in 1/10 volume of salinesolution at 4° C. The OD₆₀₀ was used to calculate the number of cellunits/mL [CU/mL] as previously described (Biswas et al. (2001) InfectImmun 69:7029-7038). Ten CU were transferred to a sterile 1.5 mLEppendorf tube and centrifuged at 13000 rpm for 1 minute at 4° C.Samples were incubated in lysis buffer (50 mM Tris-HCl 6.8, RocheComplete protease inhibitors, 4 mg/mL lysozyme, 400 U/mL mutanolysin)for 30 min at 37° C. Samples were then incubated at 4° C. for 10 minutesfollowed by sonication at 4° C. for 2×15 seconds, with 15 second pausesbetween sonications. Reactions were centrifuged at 13,000 rpm for 5minutes at 4° C., and the supernatant was transferred to a new tube andrecentrifuged at 4° C. for 10 min at 13000 rpm. A sample of thissupernatant, corresponding to crude lysate, was saved for lateranalysis. The remainder was transferred to a 200 μL slurry volume ofamylose resin (New England Biolabs), which had been washed andpre-equilibrated with column wash buffer (20 mM Tris-HCl 7.4, 200 mMNaCl, 1 mM EDTA, 1 mM DTT). Samples were batch purified by incubation at4° C. for 30 minutes with gentle tapping every 5 minutes. Reactions werethen centrifuged at 6000 rpm for 1 min at 4° C. and a samplecorresponding to the flow-through fraction was stored for lateranalysis. The resin was washed with 3×1 mL of column wash buffer at 4°C. with a 1 minute centrifugation at 6000 rpm between washes. Boundprotein was eluted in 350 μL column wash buffer containing 25 mMmaltose. SDS sample buffer (Sambrook et al., 1989) was added and sampleswere heated to 100° C. for 10 minutes.

SDS PAGE and Western Blot

SDS PAGE was conducted using NuPAGE 4-12% gradient gels (Invitrogen)with MES running buffer as previously described (Zähner and Scott (2008)J Bacteriol 190:527-535). Proteins were transferred to nitrocellulosemembranes (Bio-Rad) using a Bio-Rad mini Trans-Blot® system withtransfer buffer (25 mM Tris 8.3, 192 mM glycine) at a constant voltageof 100V for 1 hour at 4° C. Blocking solution (3% BSA in TBS 7.6, 0.02%NaN₃) was used to block membranes and for incubation of primary andsecondary antibodies. The polyclonal anti-T3 antiserum was used at adilution of 1:250. The mouse monoclonal anti-MBP antibody, a product ofNew England Biolabs, and the mouse monoclonal anti-HA antibody (HA-7), aproduct of Sigma-Aldrich, were used at dilutions of 1:2,000. Goatanti-mouse and goat anti-rabbit alkaline phosphatase conjugatedsecondary antibodies (Sigma-Aldrich) were used at a dilution of 1:3000.Signals were detected using a nitrotetrazolium blue (NBT),5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) detectionsystem.

Example 4 Intranasal Vaccination of Mice with Lactococcus lactisExpressing the Maltose Binding Protein on the Pilus Tip

To determine if the L. lactis bacteria comprising the pJRS9565 plasmidcould elicit an immune response to the displayed MBP protein in mice,CD1 mice were vaccinated intranasally with the MG1363/pJRS9565 (encodesMBP*) or the MG1363/pJRS9545 (vector control) bacteria. Blood samplesand lung lavage fluids were obtained from the mice. Anti-MBP IgG or IgAantibodies in the fluids were measured using an ELISA. As seen in FIGS.19 and 20, mice that were vaccinated with the plasmid expressing theMBP-Cpa fusion protein had developed an immune response to the displayedMBP. Therefore, polypeptides displayed on the tips of bacterial pili areeffective at mounting an immune response in mice, demonstrating theutility of such methods for the development of vaccines to variousantigens.

Materials and Methods for Experimental Example 4 Mouse Immunization

Cells (MG1363/pJRS9545 or MG1363/pJRS9565) grown at 30° C. in M17 withglucose containing 100 μg/ml spectinomycin, were washed and resuspendedin PBS to give 5×10⁷ cfu/ml. Female CD1 mice were vaccinatedintranasally by administration of 20 μA of cell suspension (10⁹ CFU).The mice were vaccinated every 10 days with a dose of 10⁹ CFU for threeconsecutive days, (i.e., the animals were vaccinated on days 1, 2, 3,14, 15, 16, and on days 27, 28, and 29). Blood samples were collectedevery 10 days (on days 1, 14, 27, and 39). The mice were sacrificed onthe 39th day, and lung lavage fluids were obtained post mortem byinserting a nylon cannula into the exposed trachea, which was tied inplace. A 1.0 ml syringe was used to inject and withdraw 1 ml of 0.9%sodium chloride solution three times, the supernatants were then storedat −80° C.

ELISA Detection of Antigen-Specific Antibodies in Serum and Lung Lavage

A 96-well EIA/RIA microplate (Costar, Corning Inc.) was coated overnightat 4° C. with 100 ng of MBP per well. The coated plate was blocked with5% soy milk in PBS-Tween to prevent nonspecific binding. Sera (1:50dilution) or lung fluid was reacted with the coated wells. Antibodyproduction was detected by using anti-mouse IgG or anti-mouse IgAsecondary antibodies coupled to alkaline phosphatase (Sigma). Absorbancewas measured at 405 nm after 45 min following the addition ofp-nitrophenyl phosphate hexahydrate disodium salt (pNPP) tabletsdissolved in diethanolamine buffer solution (KPL). The values werecorrected for background by subtracting the reading obtained with seraor lung fluid of non-immunized mice.

Example 5 Development of a live Lactococcus lactis Vaccine

As model epitopes, two different domains of the protective antigensubunit of the anthrax toxin are used to provide protection againstBacillus anthracis. Domain 1′ (residues 168-258 of SEQ ID NO: 92), whichis the domain that remains at the N-terminus of the toxin following itsproteolytic cleavage by proteases ubiquitously present in host tissue isused. This domain, called “LEF domain”, is involved in binding to theother subunits of the anthrax toxin, LF (lethal factor), and EF(edematous factor). The second domain used is domain 4 (residues 596-735of SEQ ID NO: 92), called “RBD”, which is responsible for binding of thetoxin to host cell receptors. The RBD and LEF domains are antigenic asDNA vaccines, have been inserted into the influenza virus fused withinthe hemagglutinin protein, and have been shown to provide passiveprotection against the toxin (Li et al (2005) J Virol 79:10003-10012).

Two other model antigens are used that are likely to be protectiveagainst enterotoxigenic Escherichia coli (ETEC): a mutant nontoxic formof the heat labile toxin LT, and CooD, an ETEC adhesin.

The following two antigens are used separately to generate vaccines: 1)a triple LT A mutant (whose nucleotide and amino acid sequences are setforth in SEQ ID NOs: 95 and 96, respectively) is constructed (R7K, S63K,V53E) in which three residues required for toxin activity have beenchanged in ways that don't alter the protein structure (Pizza et al(1994) J Exp Med 180:2147-2153); and 2) cooD from a CS1 ETEC strain(whose nucleotide and amino acid sequences are set forth in SEQ ID NOs:89 and 90, respectively), which will be cloned into the plasmid togetherwith its chaperone gene cooB (Voegele, Sakellaris & Scott (1997) ProcNatl Acad Sci USA 94:13257-13261). Using standard recombinant DNAtechnology, a fusion protein is engineered that has the entire antigenicprotein (either LT or CooD) fused to the C terminus of the pilus tipprotein (Cpa) in a plasmid that contains all the genes needed to make T3pili from S. pyogenes. Following DNA sequence confirmation of eachplasmid construction, western blots are used to show that, inEscherichia coli, the guest protein is polymerized with T3. Each plasmidis transformed into L. lactis and a western blot is used to identify thepresence of the guest antigen in polymerized pili. Whole cell dotimmunoblots is used to confirm surface localization in L. lactis of theguest antigen.

Each of the two L. lactis strains are introduced intraperitoneally andintranasally into mice. Serum is collected and tested in the ELISA assayfor IgG and IgA reactivity with LT or CooD. Mice are sacrificed and IgAassayed in nasal lavage, bronchio-alveolar lavage, and gut lavage fluid.

Upon detection of antibody, the ability of the anti-LT to neutralizetoxicity of whole LT-producing ETEC bacteria or anti-CooD to preventadherence of CS1 ETEC bacteria is determined.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended embodiments.

1. A method for producing a Gram-positive bacterium having at least onepolypeptide of interest covalently attached to the tip of at least onepilus, wherein said method comprises: a) introducing into saidGram-positive bacterium a polynucleotide comprising a nucleotidesequence that encodes a chimeric polypeptide, said chimeric polypeptidecomprising said polypeptide of interest and a Streptococcus pyogenespilus tip protein, active variant or active fragment thereof, whereinsaid pilus tip protein or active variant or fragment thereof comprises acell wall sorting signal (CWSS), and said pilus tip protein or fragmentthereof is carboxyl to said polypeptide of interest, and wherein saidGram-positive bacterium expresses a tip sortase and a pilus shaftpolypeptide; and b) growing said Gram-positive bacterium underconditions wherein said chimeric polypeptide is expressed and said pilusis formed. 2.-3. (canceled)
 4. The method of claim 1, wherein saidStreptococcus pyogenes pilus tip protein is selected from the groupconsisting of Cpa, protein F1, Spy0130, FctX, and FctB.
 5. The method ofclaim 1, wherein said active variant of said Streptococcus pyogenespilus tip protein has an amino acid sequence having at least 80%sequence identity to SEQ ID NO:
 3. 6. (canceled)
 7. The method of claim1, wherein said active fragment of said Streptococcus pyogenes pilus tipprotein has an amino acid sequence having at least 80% sequence identityto SEQ ID NO:
 6. 8.-10. (canceled)
 11. The method of claim 1, whereinsaid CWSS comprises: a) a CWSS motif, wherein said CWSS motif has anamino acid sequence of X₁X₂PTG, wherein X₁ and X₂ is any amino acid; b)a substantially hydrophobic domain carboxyl to said CWSS motif; and c) acharged tail region carboxyl to said substantially hydrophobic domain.12.-17. (canceled)
 18. The method of claim 1, wherein said tip sortasehas an amino acid sequence having at least 80% sequence identity to SEQID NO:
 7. 19.-22. (canceled)
 23. The method of claim 1, wherein saidpilus shaft polypeptide has an amino acid sequence having at least 80%sequence identity to SEQ ID NO:
 12. 24. (canceled)
 25. The method ofclaim 1, wherein said Gram-positive bacterium further expresses a pilinchaperone polypeptide. 26.-28. (canceled)
 29. The method of claim 25,wherein said pilin chaperone polypeptide has an amino sequence having atleast 80% sequence identity to SEQ ID NO:
 14. 30. (canceled)
 31. Themethod of claim 1, wherein said nucleotide sequence encoding saidchimeric polypeptide further comprises a nucleotide sequence encoding anamino terminal signal sequence.
 32. The method of claim 1, wherein saidGram-positive bacterium belongs to a genus selected from the groupconsisting of Actinomyces, Bacillus, Bifidobacterium, Cellulomonas,Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Lactococcus,Micrococcus, Mycobactenum, Nocardia, Staphylococcus, Streptococcus, andStreptomyces. 33.-34. (canceled)
 35. The method of claim 1, wherein saidpolypeptide of interest is selected from the group consisting of anantigen, an enzyme, a biosorbent, or an antibody or fragment thereof.36. (canceled)
 37. A Gram-positive bacterium comprising at least onepolypeptide of interest covalently attached to the tip of at least onepilus, wherein said polypeptide of interest is amino terminal to aStreptococcus pyogenes pilus tip protein or an active variant orfragment thereof, wherein said pilus tip protein or active variant oractive fragment thereof comprises a cleaved cell wall sorting signal(CWSS) motif. 38.-48. (canceled)
 49. The Gram-positive bacterium ofclaim 37, wherein said cleaved CWSS motif has an amino acid sequence ofX₁X₂PT, wherein said X₁ is any amino acid except leucine, and wherein X₂is any amino acid except proline. 50.-52. (canceled)
 53. The method ofclaim 37, wherein said pilus comprises a pilus shaft polypeptide,wherein said pilus shaft polypeptide has an amino acid sequence havingat least 80% sequence identity to SEQ ID NO:
 12. 54.-55. (canceled) 56.The Gram-positive bacterium of claim 37, wherein said polypeptide ofinterest is selected from the group consisting of an antigen, an enzyme,a biosorbent, or an antibody or fragment thereof.
 57. A method forinducing an immunological response comprising administering to a subjecta composition comprising a Gram-positive bacterium of claim 37, whereinsaid polypeptide of interest comprises an antigen.
 58. The method ofclaim 57, wherein said Gram-positive bacterium comprises an attenuatedpathogenic bacterium or a non-pathogenic bacterium.
 59. The method ofclaim 57, wherein said Gram-positive bacterium is selected from thegroup consisting of Streptococcus gordinii, Lactococcus lactis,Staphylococcus xylosus, and Staphylococcus carnosus.